Anti-cd73 antibodies and methods of use thereof

ABSTRACT

The instant disclosure provides antibodies that specifically bind to CD73 (e.g., human CD73) and antagonize CD73 function. Also provided are anti-CD73 antibodies that further comprise a TGFβ-binding moiety or a VEGF-binding moiety. The instant disclosure additionally provides pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 16/296,532, filed Mar. 8,2019, which claims priority to U.S. Provisional Patent Application Ser.No. 62/640,850, filed Mar. 9, 2018, the entire disclosure of which ishereby incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ST.26XML (Name: 199345_SL.xml; size 261,659 bytes; Created on May 15, 2023)is incorporated herein by reference in its entirety.

1. FIELD

The instant disclosure relates to antibodies that specifically bind toCD73 (e.g., human CD73) and methods of using the same.

2. BACKGROUND

CD73, also known as ecto-5′-nucleotidase, ecto-5′-NT, 5′-NT, and NT5E,is a glycosylphosphatidylinisotol (GPI)-linked cell surface enzyme. Theenzymatic activity of CD73 catalyzes hydrolysis of extracellularadenosine monophosphate (AMP) into adenosine. This activity, along withthe activity of CD39 to catalyze hydrolysis of extracellular adenosinetriphosphate (ATP) into AMP, constitutes a major adenosine-generatingsystem in the extracellular space. Adenosine is a ligand of a number ofG protein-coupled receptors (GPCRs), and is implicated in immune escape,angiogenesis, and tumor motility. CD73 is broadly expressed in manytissues, and has been found to be upregulated in late-stage primary andmetastatic tumors. Studies in animal models have shown that blockade ofCD73 activity using an anti-CD73 antibody suppresses tumor growth andprolongs survival by promoting anti-tumor adaptive immunity (Forte etal. (2012) J Immunol. 189(5):2226-33).

Transforming growth factor-β (TGFβ) is a pleiotropic cytokine that isexpressed at elevated levels in late-stage primary and metastatictumors, and is associated with proliferation, invasion, metastasis, andangiogenesis of tumor cells. TGFβ has also been shown to promote theexpansion of immunosuppressive immune populations, such as regulatory Tcells (Tregs) and myeloid-derived suppressor cells (MDSCs). See Wang etal. (2017) Front Immunol. 8: 1934.

Given the apparent role of human CD73 and TGFβ in modulating immuneresponses to tumors, therapeutic agents designed to antagonize CD73activity, or both CD73 activity and TGFβ signaling, hold great promisefor cancer treatment.

3. SUMMARY

The instant disclosure provides antibodies that specifically bind toCD73 (e.g., human CD73) and antagonize CD73 function, e.g., theenzymatic activity to promote AMP conversion to adenosine. Also providedare pharmaceutical compositions comprising these antibodies, nucleicacids encoding these antibodies, expression vectors and host cells formaking these antibodies, and methods of treating a subject using theseantibodies. The antibodies disclosed herein are particularly useful forboosting immune responses to a tumor antigen, and hence, are useful fortreating cancer in a subject.

Accordingly, in one aspect, the instant disclosure provides an isolatedantibody that specifically binds to human CD73, the antibody comprisinga heavy chain variable region comprising complementarity determiningregions (CDRs) CDRH1, CDRH2, and CDRH3, and a light chain variableregion comprising complementarity determining regions CDRL1, CDRL2, andCDRL3, wherein:

-   -   (a) CDRH1 comprises the amino acid sequence of X₁X₂WX₃X₄(SEQ ID        NO: 13), wherein        -   X₁ is S or N;        -   X₂ is S or Y;        -   X₃ is I or M; and        -   X₄ is N or H;    -   (b) CDRH2 comprises the amino acid sequence of        X₁IYPRX₂X₃DTNYX₄X₅KFKX₆ (SEQ ID NO: 14), wherein        -   X₁ is R or T;        -   X₂ is N, A, or S;        -   X₃ is G or S;        -   X₄ is N, A, or S;        -   X₅ is G or Q; and        -   X₆ is D or G;    -   (c) CDRH3 comprises the amino acid sequence of LLDYSMDY (SEQ ID        NO: 7);    -   (d) CDRL1 comprises the amino acid sequence of RASQDISX₁X₂LN        (SEQ ID NO: 16), wherein        -   X₁ is N or I; and        -   X₂ is Y or S;    -   (e) CDRL2 comprises the amino acid sequence of YTSRLHS (SEQ ID        NO: 10); and/or    -   (f) CDRL3 comprises the amino acid sequence of QQGNTLPXT (SEQ ID        NO: 17), wherein:        -   X is L or W.

In certain embodiments, CDRH1 comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1 and 2. In certainembodiments, CDRH2 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 3-6. In certain embodiments, CDRL1comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 8 and 9. In certain embodiments, CDRL3 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 11 and12.

In certain embodiments,

-   -   (a) CDRH1 comprises the amino acid sequence of SSWIN (SEQ ID NO:        2);    -   (b) CDRH2 comprises the amino acid sequence of        RIYPRX₁GDTNYX₂GKFKD (SEQ ID NO: 15), wherein        -   X₁ is N, A, or S; and        -   X₂ is N, A, or S;    -   (c) CDRL1 comprises the amino acid sequence of RASQDISX₁X₂LN        (SEQ ID NO: 16), wherein        -   X₁ is N or I; and        -   X₂ is Y or S; and/or    -   (d) CDRL3 comprises the amino acid sequence of QQGNTLPLT (SEQ ID        NO: 12).

In certain embodiments, CDRH2 comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 4-6. In certain embodiments,CDRL1 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8 and 9.

In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the CDRH1,CDRH2, and CDRH3 amino acid sequences, respectively, set forth in SEQ IDNOs: 2, 4, and 7; 2, 5, and 7; 1, 3, and 7; or 2, 6, and 7. In certainembodiments, CDRL1, CDRL2, and CDRL3 comprise the CDRL1, CDRL2, andCDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs: 8,10, and 12; 9, 10, and 12; or 8, 10, and 11. In certain embodiments,CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences, respectively, setforth in SEQ ID NOs: 2, 4, 7, 8, 10, and 12; 2, 5, 7, 8, 10, and 12; 1,3, 7, 8, 10, and 11; 2, 4, 7, 8, 10, and 11; 2, 4, 7, 9, 10, and 12; 2,6, 7, 8, 10, and 12; 2, 5, 7, 9, 10, and 12; or 2, 6, 7, 9, 10, and 12.In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acidsequences, respectively, set forth in SEQ ID NOs: 2, 4, 7, 8, 10, and12; or 2, 5, 7, 8, 10, and 12.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, the antibody comprising a heavychain variable region comprising CDRs CDRH1, CDRH2, and CDRH3, and alight chain variable region comprising complementarity determiningregions CDRL1, CDRL2, and CDRL3, wherein CDRH3 comprises the amino acidsequence of LLDYSMDY (SEQ ID NO: 7); and/or CDRL3 comprises the aminoacid sequence of QQGNTLPLT (SEQ ID NO: 12) or QQGNTLPWT (SEQ ID NO: 11).

In certain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 31. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 19-21 and23-30. In certain embodiments, the heavy chain variable region comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:19-21 and 23-30. In certain embodiments, the antibody comprises a heavychain variable region comprising an amino acid sequence at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 23-30. In certain embodiments, the heavy chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 23-30. In certain embodiments, the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 24 or27. In certain embodiments, the antibody comprises a heavy chainvariable region comprising an amino acid sequence derived from a humangermline sequence as set forth in SEQ ID NO: 22.

In certain embodiments, the antibody comprises a light chain variableregion comprising an amino acid sequence of SEQ ID NO: 41 or 42. Incertain embodiments, the antibody comprises a light chain variableregion comprising an amino acid sequence at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to anamino acid sequence selected from the group consisting of SEQ ID NOs:33-35 and 37-40. In certain embodiments, the light chain variable regioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 33-35 and 37-40. In certain embodiments, the antibodycomprises a light chain variable region comprising an amino acidsequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 37-40. In certain embodiments, the lightchain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 37-40. In certain embodiments, the lightchain variable region comprises the amino acid sequence of SEQ ID NO:37. In certain embodiments, the antibody comprises a light chainvariable region comprising an amino acid sequence derived from a humangermline sequence as set forth in SEQ ID NO: 36.

In certain embodiments, the heavy chain variable region and the lightchain variable region, respectively, comprise the amino acid sequencesset forth in SEQ ID NOs: 24 and 37; 27 and 37; 19 and 33; 20 and 34; 21and 35; 23 and 37; 23 and 38; 24 and 38; 25 and 37; 26 and 37; 28 and37; 25 and 38; 26 and 38; 27 and 38; 28 and 38; 29 and 37; 30 and 37; 23and 39; or 23 and 40. In certain embodiments, the heavy chain variableregion and the light chain variable region, respectively, comprise theamino acid sequences set forth in SEQ ID NOs: 24 and 37; or 27 and 37.

In certain embodiments, the antibody comprises a heavy chain variableregion comprising an amino acid sequence at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNOs: 27, and/or a light chain variable region comprising an amino acidsequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NOs: 37

In certain embodiments, the antibody comprises a heavy chain comprisingan amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 85, and/or alight chain comprising an amino acid sequence at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NOs: 97.

In certain embodiments, the antibody comprises a heavy chain comprisingan amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 115, and/or alight chain comprising an amino acid sequence at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NOs: 97.

In certain embodiments, the antibody comprises a heavy chain constantregion selected from the group consisting of human IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂. In certain embodiments, the heavy chain constantregion is IgG₁. In certain embodiments, the amino acid sequence of IgG₁comprises a N297A mutation, numbered according to the EU numberingsystem. In certain embodiments, the heavy chain constant regioncomprises the amino acid sequence of SEQ ID NO: 50, 49, 46, 45, 55, or56. In certain embodiments, the antibody comprises a heavy chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 63-88.

In certain embodiments, the antibody comprises a light chain constantregion selected from the group consisting of human Igκ and Igλ. Incertain embodiments, the light chain constant region is Igκ. In certainembodiments, the light chain constant region comprises the amino acidsequence of SEQ ID NO: 93 or 89. In certain embodiments, the antibodycomprises a light chain comprising the amino acid sequence selected fromthe group consisting of SEQ ID NO: 92 and 94-98.

In certain embodiments, the antibody comprises a heavy chain and a lightchain comprising the amino acid sequences set forth in any row of Table4, preferably the amino acid sequence selected from the group consistingof SEQ ID NOs: 79 and 97; 85 and 97; 77 and 97; 80 and 97; 86 and 97; 64and 92; 70 and 96; or 78 and 97, respectively.

In certain embodiments reciting any one of SEQ ID NOs: 19, 20, and65-68, the X in any one of SEQ ID NOs: 19, 20, and 65-68 is glutamine.In certain embodiments reciting any one of SEQ ID NOs: 21, 23-30, 63,64, 69-88, the X in any one of SEQ ID NOs: 21, 23-30, 63, 64, 69-88 isglutamate. In certain embodiments reciting any one of SEQ ID NOs: 19-21,23-30, and 63-88, the X in any one of SEQ ID NOs: 19-21, 23-30, and63-88 is pyroglutamate.

In another aspect, the instant disclosure provides an isolated antibodythat binds to the same epitope of human CD73 as the antibody of any oneof the preceding claims.

In certain embodiments of any one of the foregoing aspects, the antibodyis a humanized antibody. In certain embodiments, the antibody isantagonistic to human CD73. In certain embodiments, the antibodydeactivates, reduces, or inhibits an activity of human CD73. In certainembodiments, the antibody attenuates the ability of human CD73 toconvert adenosine monophosphate to adenosine. In certain embodiments,the antibody is internalized upon binding to cells expressing humanCD73.

In certain embodiments, the isolated antibody further comprises aTGFβ-binding moiety. In certain embodiments, the TGFβ-binding moiety islinked to the heavy chain variable region. In certain embodiments, theTGFβ-binding moiety is linked to the heavy chain variable region via apeptide linker. In certain embodiments, the isolated antibody furthercomprises a TGFβ-binding moiety linked to the heavy chain constantregion. In certain embodiments, the TGFβ-binding moiety is linked to theC-terminal residue of the heavy chain constant region. In certainembodiments, the N-terminal residue of the TGFβ-binding moiety is linkedto the C-terminal residue of the heavy chain constant region via apeptide linker. In certain embodiments, the peptide linker comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:103-108.

In certain embodiments, the TGFβ-binding moiety specifically binds tohuman TGFβ. In certain embodiments, the TGFβ-binding moiety comprises anextracellular domain of a human TGFβ receptor. In certain embodiments,the human TGFβ receptor is selected from the group consisting of humanTGFβR1, TGFβR2, and TGFβR3. In certain embodiments, the human TGFβreceptor is human TGFβR2. In certain embodiments, the TGFβ-bindingmoiety comprises an amino acid sequence at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequenceselected from the group consisting of SEQ ID NOs: 109-112. In certainembodiments, the TGFβ-binding moiety consists of an amino acid sequenceat least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a sequence selected from the group consisting of SEQ IDNOs: 109-112. In certain embodiments, the TGFβ-binding moiety comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:109-112, preferably SEQ ID NO: 111. In certain embodiments, theTGFβ-binding moiety consists of an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 109-112, preferably SEQ ID NO: 111.

In certain embodiments, the isolated antibody comprises a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 113-121, preferably SEQ ID NO: 114 or 115. In certainembodiments, the isolated antibody comprises a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:113-121 and a polypeptide comprising the amino acid sequence of SEQ IDNO: 92 or 97. In certain embodiments, the isolated antibody comprisestwo polypeptides each comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 113-121 and two polypeptides eachcomprising the amino acid sequence of SEQ ID NO: 92 or 97. In certainembodiments, the isolated antibody comprises two polypeptides eachcomprising the amino acid sequence of SEQ ID NO: 115 and twopolypeptides each comprising the amino acid sequence of SEQ ID NO: 97.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, the antibody comprising aTGFβ-binding moiety. In certain embodiments, the antibody isantagonistic to human CD73. In certain embodiments, the antibodydeactivates, reduces, or inhibits an activity of human CD73. In certainembodiments, the antibody attenuates the ability of human CD73 toconvert adenosine monophosphate to adenosine. In certain embodiments,the antibody, when bound to TGFβ, is antagonistic to human CD73.

In certain embodiments, the TGFβ-binding moiety specifically binds tohuman TGFβ. In certain embodiments, the TGFβ-binding moiety comprises anextracellular domain of a human TGFβ receptor. In certain embodiments,the human TGFβ receptor is selected from the group consisting of humanTGFβR1, TGFβR2, and TGFβR3. In certain embodiments, the human TGFβreceptor is human TGFβR2. In certain embodiments, the TGFβ-bindingmoiety comprises an amino acid sequence at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequenceselected from the group consisting of SEQ ID NOs: 109-112. In certainembodiments, the TGFβ-binding moiety consists of an amino acid sequenceat least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a sequence selected from the group consisting of SEQ IDNOs: 109-112. In certain embodiments, the TGFβ-binding moiety comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:109-112, preferably SEQ ID NO: 111. In certain embodiments, theTGFβ-binding moiety consists of an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 109-112, preferably SEQ ID NO: 111.

In certain embodiments, the isolated antibody further comprises aVEGF-binding moiety. In certain embodiments, the VEGF-binding moiety islinked to the heavy chain variable region. In certain embodiments, theVEGF-binding moiety is linked to the heavy chain variable region via apeptide linker. In certain embodiments, the isolated antibody furthercomprises a VEGF-binding moiety linked to the heavy chain constantregion. In certain embodiments, the VEGF-binding moiety is linked to theC-terminal residue of the heavy chain constant region. In certainembodiments, the N-terminal residue of the VEGF-binding moiety is linkedto the C-terminal residue of the heavy chain constant region via apeptide linker. In certain embodiments, the peptide linker comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:103-108.

In certain embodiments, the VEGF-binding moiety specifically binds tohuman VEGF. In certain embodiments, the VEGF-binding moiety comprises anextracellular domain of a human VEGF receptor. In certain embodiments,the human VEGF receptor is selected from the group consisting of humanVEGFR1, VEGFR2, and VEGFR3. In certain embodiments, the human VEGFreceptor is human VEGFR1. In certain embodiments, the VEGF-bindingmoiety comprises an amino acid sequence at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequenceset forth in SEQ ID NO: 122. In certain embodiments, the VEGF-bindingmoiety consists of an amino acid sequence at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequenceset forth in SEQ ID NO: 122. In certain embodiments, the VEGF-bindingmoiety comprises the sequence set forth in SEQ ID NO: 122. In certainembodiments, the VEGF-binding moiety consists of the sequence set forthin SEQ ID NO: 122.

In certain embodiments, the isolated antibody comprises a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 123-127. In certain embodiments, the antibody comprises apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 123-127 and a polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 92, 128,and 97. In certain embodiments, the antibody comprises two polypeptidescomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 123-127 and two polypeptides comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 92, 128, and97.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, the antibody comprising aVEGF-binding moiety. In certain embodiments, the antibody isantagonistic to human CD73. In certain embodiments, the antibodydeactivates, reduces, or inhibits an activity of human CD73. In certainembodiments, the antibody attenuates the ability of human CD73 toconvert adenosine monophosphate to adenosine. In certain embodiments,the antibody, when bound to VEGF, is antagonistic to human CD73.

In certain embodiments, the VEGF-binding moiety specifically binds tohuman VEGF. In certain embodiments, the VEGF-binding moiety comprises anextracellular domain of a human VEGF receptor. In certain embodiments,the human VEGF receptor is selected from the group consisting of humanVEGFR1, VEGFR2, and VEGFR3. In certain embodiments, the human VEGFreceptor is human VEGFR1. In certain embodiments, the VEGF-bindingmoiety comprises an amino acid sequence at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequenceset forth in SEQ ID NO: 122. In certain embodiments, the VEGF-bindingmoiety consists of an amino acid sequence at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequenceset forth in SEQ ID NO: 122. In certain embodiments, the VEGF-bindingmoiety comprises the amino acid sequence as set forth in SEQ ID NO: 122.In certain embodiments, the VEGF-binding moiety consists of the aminoacid sequence as set forth in SEQ ID NO: 122.

In certain embodiments of any one of the foregoing aspects, the antibodyfurther comprises a conjugated cytotoxic agent, cytostatic agent, toxin,radionuclide, or detectable label.

In certain embodiments of any one of the foregoing aspects, the antibodybinds to an epitope located within a region of human CD73, the aminoacid sequence of the region consisting of the amino acid sequence of SEQID NOs: 90 and/or 91.

In certain embodiments, the antibody binds to at least one residue inthe amino acid sequence of SEQ ID NOs: 90 and/or 91.

In certain embodiments, the antibody binds to one or more amino acidresidues of human CD73 selected from the group consisting of Y158, Y161,P165, or D168, numbered according to the amino acid sequence of SEQ IDNO: 99.

In certain embodiments, the binding affinity of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 133 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In certain embodiments, the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 133 issubstantially reduced (e.g., reduced by at least 30%, 40%, 50%, 60%,70%, 80% or 90%) relative to the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 141.

In certain embodiments, the binding affinity of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 134 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In certain embodiments, the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 134 issubstantially reduced (e.g., reduced by at least 30%, 40%, 50%, 60%,70%, 80% or 90%) relative to the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 141.

In certain embodiments, the antibody binds to amino acid residue Y158 ofhuman CD73, numbered according to the amino acid sequence of SEQ ID NO:99.

In certain embodiments, the binding affinity of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 59 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In certain embodiments, the antibody binds to amino acid residue Y161 ofhuman CD73, numbered according to the amino acid sequence of SEQ ID NO:99.

In certain embodiments, the binding affinity of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 60 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In certain embodiments, the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 60 issubstantially reduced (e.g., reduced by at least 30%, 40%, 50%, 60%,70%, 80% or 90%) relative to the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 141.

In certain embodiments, the antibody binds to amino acid residue P165 ofhuman CD73, numbered according to the amino acid sequence of SEQ ID NO:99.

In certain embodiments, the binding affinity of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 139 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In certain embodiments, the antibody binds to amino acid residue D168 ofhuman CD73, numbered according to the amino acid sequence of SEQ ID NO:99.

In certain embodiments, the binding affinity of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 140 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In certain embodiments, the binding affinity of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 135 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In certain embodiments, the binding affinity of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 136 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In certain embodiments, the binding affinity of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 142 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141, and wherein the binding affinity of theantibody for a protein comprising the amino acid sequence of SEQ ID NO:137 is not substantially reduced (e.g., not reduced by more than 4times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times)relative to the binding affinity of the antibody for a proteincomprising the amino acid sequence of SEQ ID NO: 141.

In certain embodiments, the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 142 issubstantially reduced (e.g., reduced by at least 30%, 40%, 50%, 60%,70%, 80% or 90%) relative to the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 141, andwherein the maximal binding of the antibody for a protein comprising theamino acid sequence of SEQ ID NO: 137 is not substantially reduced(e.g., not reduced by more than 30%, 40%, 50%, 60%, 70%, 80% or 90%)relative to the maximal binding of the antibody for a protein comprisingthe amino acid sequence of SEQ ID NO: 141.

In certain embodiments, the binding affinity of the antibody to aprotein comprising the amino acid sequence of SEQ ID NO: 142 issubstantially reduced (e.g., reduced by at least 4 times, 5 times, 6times, 7 times, 8 times, 9 times, or 10 times) relative to the bindingaffinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141, and wherein the binding affinity of theantibody for a protein comprising the amino acid sequence of SEQ ID NO:138 is not substantially reduced (e.g., not reduced by more than 4times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times)relative to the binding affinity of the antibody for a proteincomprising the amino acid sequence of SEQ ID NO: 141.

In certain embodiments, the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 142 issubstantially reduced (e.g., reduced by at least 30%, 40%, 50%, 60%,70%, 80% or 90%) relative to the maximal binding of the antibody for aprotein comprising the amino acid sequence of SEQ ID NO: 141, andwherein the maximal binding of the antibody for a protein comprising theamino acid sequence of SEQ ID NO: 138 is not substantially reduced(e.g., not reduced by more than 30%, 40%, 50%, 60%, 70%, 80% or 90%)relative to the maximal binding of the antibody for a protein comprisingthe amino acid sequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the antibody binds to anepitope located within a region of human CD73, the amino acid sequenceof the region consisting of the amino acid sequence of SEQ ID NOs: 90and/or 91.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the antibody binds to atleast one residue in the amino acid sequence of SEQ ID NOs: 90 and/or91.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the antibody binds to oneor more amino acid residues of human CD73 selected from the groupconsisting of Y158, Y161, P165, or D168, numbered according to the aminoacid sequence of SEQ ID NO: 99.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 133 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the maximal binding ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 133 is substantially reduced (e.g., reduced by at least 30%, 40%,50%, 60%, 70%, 80% or 90%) relative to the maximal binding of theantibody for a protein comprising the amino acid sequence of SEQ ID NO:141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 134 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the maximal binding ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 134 is substantially reduced (e.g., reduced by at least 30%, 40%,50%, 60%, 70%, 80% or 90%) relative to the maximal binding of theantibody for a protein comprising the amino acid sequence of SEQ ID NO:141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the antibody binds toamino acid residue Y158 of human CD73, numbered according to the aminoacid sequence of SEQ ID NO: 99.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 59 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the antibody binds toamino acid residue Y161 of human CD73, numbered according to the aminoacid sequence of SEQ ID NO: 99.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 60 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the maximal binding ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 60 is substantially reduced (e.g., reduced by at least 30%, 40%,50%, 60%, 70%, 80% or 90%) relative to the maximal binding of theantibody for a protein comprising the amino acid sequence of SEQ ID NO:141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the antibody binds toamino acid residue P165 of human CD73, numbered according to the aminoacid sequence of SEQ ID NO: 99.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 139 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the antibody binds toamino acid residue D168 of human CD73, numbered according to the aminoacid sequence of SEQ ID NO: 99.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 140 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 135 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 136 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 142 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141, and wherein the binding affinity of theantibody for a protein comprising the amino acid sequence of SEQ ID NO:137 is not substantially reduced (e.g., not reduced by more than 4times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times)relative to the binding affinity of the antibody for a proteincomprising the amino acid sequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the maximal binding ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 142 is substantially reduced (e.g., reduced by at least 30%, 40%,50%, 60%, 70%, 80% or 90%) relative to the maximal binding of theantibody for a protein comprising the amino acid sequence of SEQ ID NO:141, and wherein the maximal binding of the antibody for a proteincomprising the amino acid sequence of SEQ ID NO: 137 is notsubstantially reduced (e.g., not reduced by more than 30%, 40%, 50%,60%, 70%, 80% or 90%) relative to the maximal binding of the antibodyfor a protein comprising the amino acid sequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the binding affinity ofthe antibody to a protein comprising the amino acid sequence of SEQ IDNO: 142 is substantially reduced (e.g., reduced by at least 4 times, 5times, 6 times, 7 times, 8 times, 9 times, or 10 times) relative to thebinding affinity of the antibody for a protein comprising the amino acidsequence of SEQ ID NO: 141, and wherein the binding affinity of theantibody for a protein comprising the amino acid sequence of SEQ ID NO:138 is not substantially reduced (e.g., not reduced by more than 4times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times)relative to the binding affinity of the antibody for a proteincomprising the amino acid sequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CD73, wherein the maximal binding ofthe antibody for a protein comprising the amino acid sequence of SEQ IDNO: 142 is substantially reduced (e.g., reduced by at least 30%, 40%,50%, 60%, 70%, 80% or 90%) relative to the maximal binding of theantibody for a protein comprising the amino acid sequence of SEQ ID NO:141, and wherein the maximal binding of the antibody for a proteincomprising the amino acid sequence of SEQ ID NO: 138 is notsubstantially reduced (e.g., not reduced by more than 30%, 40%, 50%,60%, 70%, 80% or 90%) relative to the maximal binding of the antibodyfor a protein comprising the amino acid sequence of SEQ ID NO: 141.

In another aspect, the instant disclosure provides a compound ormolecule comprising a CD73-binding moiety and a TGFβ-binding moiety.

In another aspect, the instant disclosure provides a pharmaceuticalcomposition comprising the antibody disclosed herein and apharmaceutically acceptable carrier or excipient.

In another aspect, the instant disclosure provides an isolatedpolynucleotide encoding a polypeptide of the antibody disclosed herein.In certain embodiments, the polynucleotide comprises cDNA or mRNA. Inanother aspect, the instant disclosure provides a vector comprising thepolynucleotide. In certain embodiments, the vector is a plasmid vectoror a viral vector. In another aspect, the instant disclosure provides arecombinant host cell comprising the polynucleotide or the vector. Inanother aspect, the instant disclosure provides a lipid nanoparticlecomprising the polynucleotide or the vector. In another aspect, theinstant disclosure provides a method of producing an antibody, themethod comprising culturing the host cell under suitable conditions sothat the polynucleotide is expressed and the antibody is produced. Inanother aspect, the instant disclosure provides a pharmaceuticalcomposition comprising the antibody, polynucleotide, vector, or lipidnanoparticle and a pharmaceutically acceptable carrier or excipient.

In another aspect, the instant disclosure provides a method of treatingcancer in a subject, the method comprising administering to the subjectan effective amount of the antibody, polynucleotide, vector, lipidnanoparticle, or pharmaceutical composition disclosed herein. In anotheraspect, the instant disclosure provides a method of inhibiting cancermetastasis in a subject, the method comprising administering to thesubject an effective amount of the antibody, polynucleotide, vector,lipid nanoparticle, or pharmaceutical composition disclosed herein. Incertain embodiments, the antibody, polynucleotide, vector, lipidnanoparticle, or pharmaceutical composition is administeredintravenously intravenously, intratumorally, subcutaneously,intradermally, intramuscularly, intravesically, intracranially,intracavitary or intraventricularly. In certain embodiments, theantibody, polynucleotide, vector, lipid nanoparticle, or pharmaceuticalcomposition is administered intratumorally.

In certain embodiments, the method further comprises administering oneor more additional therapeutic agent to the subject. In certainembodiments, the antibody, polynucleotide, vector, lipid nanoparticle,or pharmaceutical composition, and the one or more additionaltherapeutic agents are co-administered according to the same schedule(e.g., co-administered at the same time intervals). In certainembodiments, the antibody, polynucleotide, vector, lipid nanoparticle,or pharmaceutical composition, and the one or more additionaltherapeutic agents are co-administered according to different schedules(e.g., co-administered at different time intervals). In certainembodiments, the antibody, polynucleotide, vector, lipid nanoparticle,or pharmaceutical composition is co-administered with one or moreadditional therapeutic agents comprising an activator or agonist of afms related tyrosine kinase 3 (FLT3; CD135) receptor, a toll-likereceptor (TLR) or a stimulator of interferon genes (STING) receptor. Incertain embodiments, the TLR agonist or activator is selected from thegroup consisting of a TLR2 agonist, a TLR3 agonist, a TLR7 agonist, aTLR8 agonist and a TLR9 agonist. In certain embodiments, the STINGreceptor agonist or activator is selected from the group consisting ofADU-S100 (MIW-815), SB-11285, MK-1454, SR-8291, AdVCA0848, GSK-532,SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthenone-4-acetic acid (DMXAA),cyclic-GAMP (cGAMP) and cyclic-di-AMP. In certain embodiments, theantibody, polynucleotide, vector, lipid nanoparticle, or pharmaceuticalcomposition is co-administered with one or more additional therapeuticagents comprising an inhibitor or antagonist of: protein tyrosinephosphatase, non-receptor type 11 (PTPN11 or SHP2), myeloid cellleukemia sequence 1 (MCL1) apoptosis regulator; mitogen-activatedprotein kinase kinase kinase kinase 1 (MAP4K1) (also calledHematopoietic Progenitor Kinase 1 (HPK1)); diacylglycerol kinase alpha(DGKA, DAGK, DAGK1 or DGK-alpha); 5′-nucleotidase ecto (NT5E or CD73);transforming growth factor beta 1 (TGFB1 or TGFβ); heme oxygenase 1(HMOX1, HO-1 or HO1); vascular endothelial growth factor A (VEGFA orVEGF); erb-b2 receptor tyrosine kinase 2 (ERBB2 HER2, HER2/neu orCD340); epidermal growth factor receptor (EGFR, ERBB, ERBB1 or HER1);ALK receptor tyrosine kinase (ALK, CD246); poly(ADP-ribose) polymerase 1(PARP1 or PARP); cyclin dependent kinase 4 (CDK4); cyclin dependentkinase 6 (CDK6); C—C motif chemokine receptor 8 (CCR8, CDw198); CD274molecule (CD274, PDL1 or PD-L1); programmed cell death 1 (PDCD1, PD1 orPD-1); and/or cytotoxic T-lymphocyte associated protein 4 (CTLA4,CTLA-4, CD152). In certain embodiments, the inhibitor comprises anantigen binding molecule, an antibody or an antigen-binding fragmentthereof. In certain embodiments, the inhibitor comprises a small organicmolecule. In certain embodiments, the inhibitor of MCL1 is selected fromthe group consisting of AMG-176, AMG-397, 5-64315, AZD-5991, 483-LM, A1210477, UMI-77 and JKY-5-037. In certain embodiments, the inhibitor ofPTPN11 or SHP2 is selected from the group consisting of TNO155(SHP-099), RMC-4550, JAB-3068 and RMC-4630.

In certain embodiments, the one or more additional therapeutic agent isa chemotherapeutic, an anti-neoplastic, a radiotherapeutic, or acheckpoint targeting agent. In certain embodiments, the one or moreanti-neoplastic or chemotherapeutic agents are selected from the groupconsisting of a nucleoside analog (e.g., 5-fluorouracil, gemcitabine,cytarabine), a taxane (e.g., paclitaxel, nab-paclitaxel, docetaxel,cabazitaxel), a platinum coordination complex (cisplatin, carboplatin,oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin,picoplatin, satraplatin, dicycloplatin, eptaplatin, lobaplatin,miriplatin), a dihydrofolate reductase (DHFR) inhibitor (e.g.,methotrexate, trimetrexate, pemetrexed), a topoisomerase inhibitor(e.g., doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin, irinotecan, mitoxantrone, pixantrone, sobuzoxane,topotecan, irinotecan, MM-398 (liposomal irinotecan), vosaroxin andGPX-150, aldoxorubicin, AR-67, mavelertinib, AST-2818, avitinib(ACEA-0010), irofulven (MGI-114)), an alkylating agent (e.g., a nitrogenmustard (e.g., cyclophosphamide, chlormethine, uramustine or uracilmustard, melphalan, chlorambucil, ifosfamide, bendamustine,temozolomide, carmustine), a nitrosourea (e.g., carmustine, lomustine,streptozocin), an alkyl sulfonate (e.g., busulfan)), and mixturesthereof.

In certain embodiments, the additional therapeutic agent is a checkpointtargeting agent. In certain embodiments, the checkpoint targeting agentis selected from the group consisting of an antagonist anti-PD-1antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2antibody, an antagonist anti-CTLA-4 antibody, an antagonist anti-TIM-3antibody, an antagonist anti-LAG-3 antibody, an antagonist anti-CEACAM1antibody, an agonist anti-GITR antibody, an antagonist anti-TIGITantibody, an antagonist anti-VISTA antibody, an agonist anti-CD137antibody, and an agonist anti-OX40 antibody. In certain embodiments, theadditional therapeutic agent is an anti-PD-1 antibody, optionallywherein the anti-PD-1 antibody is pembrolizumab or nivolumab. In certainembodiments, the additional therapeutic agent is an inhibitor ofindoleamine-2,3-dioxygenase (IDO). In certain embodiments, the inhibitoris selected from the group consisting of epacadostat, F001287,indoximod, and NLG919. In certain embodiments, the inhibitor isepacadostat. In certain embodiments, the additional therapeutic agent isa vaccine. In certain embodiments, the vaccine comprises a heat shockprotein peptide complex (HSPPC) comprising a heat shock proteincomplexed with an antigenic peptide. In certain embodiments, the heatshock protein is hsc70 and is complexed with a tumor-associatedantigenic peptide. In certain embodiments, the heat shock protein isgp96 and is complexed with a tumor-associated antigenic peptide, whereinthe HSPPC is derived from a tumor obtained from a subject. In certainembodiments, the additional therapeutic agent is etoposide ordoxorubicin.

In another aspect, the antibody, polynucleotide, vector, lipidnanoparticle, or pharmaceutical composition is co-administered with aFOLFOX regimen, a FOLFOXIRI regimen or a FOLFIRINOX regimen. In anotheraspect, the antibody, polynucleotide, vector, lipid nanoparticle, orpharmaceutical composition is co-administered with an immunotherapy, animmunostimulatory therapy, a cellular therapy or a gene therapy.

In another aspect, the instant disclosure provides an isolated antibody,polynucleotide, vector, lipid nanoparticle, or pharmaceuticalcomposition disclosed herein for use in the treatment of cancer.

In another aspect, the instant disclosure provides use of an isolatedantibody, polynucleotide, vector, lipid nanoparticle, or pharmaceuticalcomposition disclosed herein for the preparation of a medicament fortreating cancer.

In certain embodiments of any one of the aspects of method of treatingcancer in a subject, the instant disclosure provides an isolatedantibody, polynucleotide, vector, lipid nanoparticle, or pharmaceuticalcomposition for use in the treatment of cancer, or use of an isolatedantibody, polynucleotide, vector, lipid nanoparticle, or pharmaceuticalcomposition disclosed herein for the preparation of a medicament fortreating cancer, the cancer is selected from the group consisting of asolid tumor, a hematological cancer, and a metastatic lesion. In certainembodiments, the cancer is a solid tumor. In certain embodiments, thesolid tumor is selected from the group consisting of a sarcoma, afibroblastic sarcoma, a carcinoma, and an adenocarcinoma. In certainembodiments, the cancer is a hematological cancer. In certainembodiments, the hematological cancer is selected from the groupconsisting of a leukemia, a lymphoma, and a myeloma. In certainembodiments, the cancer is selected from the group consisting of a lungcancer, a melanoma, a renal cancer, a liver cancer, a myeloma, aprostate cancer, a breast cancer, an ovarian cancer, a colorectalcancer, a pancreatic cancer, a head and neck cancer, an anal cancer, agastro-esophageal cancer, a mesothelioma, a nasopharyngeal cancer, athyroid cancer, a cervical cancer, an epithelial cancer, a peritonealcancer, a lymphoproliferative disease, an acute lymphoblastic leukemia(ALL), an acute myelogenous leukemia (AML), a chronic lymphocyticleukemia (CLL), a chronic myelogenous leukemia (CML), a chronicmyelomonocytic leukemia (CMML), a hairy cell leukemia, a B celllymphoma, a diffuse large B-cell lymphoma (DLBCL), an activated B-celllike (ABC) diffuse large B cell lymphoma, a germinal center B cell (GCB)diffuse large B cell lymphoma, a mantle cell lymphoma, a Hodgkinlymphoma, a non-Hodgkin lymphoma, a relapsed non-Hodgkin lymphoma, arefractory non-Hodgkin lymphoma, a recurrent follicular non-Hodgkinlymphoma, a Burkitt lymphoma, a small lymphocytic lymphoma, a follicularlymphoma, a lymphoplasmacytic lymphoma, and an extranodal marginal zonelymphoma. In certain embodiments, the cancer is a solid tumor in orarising from a tissue or organ selected from the group consisting of:bone (e.g., adamantinoma, aneurysmal bone cysts, angiosarcoma,chondroblastoma, chondroma, chondromyxoid fibroma, chondrosarcoma,chordoma, dedifferentiated chondrosarcoma, enchondroma, epithelioidhemangioendothelioma, fibrous dysplasia of the bone, giant cell tumourof bone, haemangiomas and related lesions, osteoblastoma,osteochondroma, osteosarcoma, osteoid osteoma, osteoma, periostealchondroma, Desmoid tumor, Ewing sarcoma); lips and oral cavity (e.g.,odontogenic ameloblastoma, oral leukoplakia, oral squamous cellcarcinoma, primary oral mucosal melanoma); salivary glands (e.g.,pleomorphic salivary gland adenoma, salivary gland adenoid cysticcarcinoma, salivary gland mucoepidermoid carcinoma, salivary glandWarthin's tumors); esophagus (e.g., Barrett's esophagus, dysplasia andadenocarcinoma); gastrointestinal tract, including stomach (e.g.,gastric adenocarcinoma, primary gastric lymphoma, gastrointestinalstromal tumors (GISTs), metastatic deposits, gastric carcinoids, gastricsarcomas, neuroendocrine carcinoma, gastric primary squamous cellcarcinoma, gastric adenoacanthomas), intestines and smooth muscle (e.g.,intravenous leiomyomatosis), colon (e.g., colorectal adenocarcinoma),rectum, anus; pancreas (e.g., serous neoplasms, including microcystic ormacrocystic serous cystadenoma, solid serous cystadenoma, VonHippel-Landau (VHL)-associated serous cystic neoplasm, serouscystadenocarcinoma, mucinous cystic neoplasms (MCN), intraductalpapillary mucinous neoplasms (IPMN), intraductal oncocytic papillaryneoplasms (IOPN), intraductal tubular neoplasms, cystic acinarneoplasms, including acinar cell cystadenoma, acinar cellcystadenocarcinoma, pancreatic adenocarcinoma, invasive pancreaticductal adenocarcinomas, including tubular adenocarcinoma, adenosquamouscarcinoma, colloid carcinoma, medullary carcinoma, hepatoid carcinoma,signet ring cell carcinoma, undifferentiated carcinoma, undifferentiatedcarcinoma with osteoclast-like giant cells, acinar cell carcinoma,neuroendocrine neoplasms, neuroendocrine microadenoma, neuroendocrinetumors (NET), neuroendocrine carcinoma (NEC), including small cell orlarge cell NEC, insulinoma, gastrinoma, glucagonoma, serotonin-producingNET, somatostatinoma, VIPoma, solid-pseudopapillary neoplasms (SPN),pancreatoblastoma); gall bladder (e.g. carcinoma of the gallbladder andextrahepatic bile ducts, intrahepatic cholangiocarcinoma);neuro-endocrine (e.g., adrenal cortical carcinoma, carcinoid tumors,phaeochromocytoma, pituitary adenomas); thyroid (e.g., anaplastic(undifferentiated) carcinoma, medullary carcinoma, oncocytic tumors,papillary carcinoma, adenocarcinoma); liver (e.g., adenoma, combinedhepatocellular and cholangiocarcinoma, fibrolamellar carcinoma,hepatoblastoma, hepatocellular carcinoma, mesenchymal, nested stromalepithelial tumor, undifferentiated carcinoma, hepatocellular carcinoma,intrahepatic cholangiocarcinoma, bile duct cystadenocarcinoma,epithelioid hemangioendothelioma, angiosarcoma, embryonal sarcoma,rhabdomyosarcoma, solitary fibrous tumor, teratoma, York sac tumor,carcinosarcoma, rhabdoid tumor); kidney (e.g., ALK-rearranged renal cellcarcinoma, chromophobe renal cell carcinoma, clear cell renal cellcarcinoma, clear cell sarcoma, metanephric adenoma, metanephricadenofibroma, mucinous tubular and spindle cell carcinoma, nephroma,nephroblastoma (Wilms tumor), papillary adenoma, papillary renal cellcarcinoma, renal oncocytoma, renal cell carcinoma, succinatedehydrogenase-deficient renal cell carcinoma, collecting ductcarcinoma); breast (e.g., invasive ductal carcinoma, including withoutlimitation, acinic cell carcinoma, adenoid cystic carcinoma, apocrinecarcinoma, cribriform carcinoma, glycogen-rich/clear cell, inflammatorycarcinoma, lipid-rich carcinoma, medullary carcinoma, metaplasticcarcinoma, micropapillary carcinoma, mucinous carcinoma, neuroendocrinecarcinoma, oncocytic carcinoma, papillary carcinoma, sebaceouscarcinoma, secretory breast carcinoma, tubular carcinoma, lobularcarcinoma, including without limitation, pleomorphic carcinoma, signetring cell carcinoma, peritoneum (e.g., mesothelioma, primary peritonealcancer)); female sex organ tissues, including ovary (e.g.,choriocarcinoma, epithelial tumors, germ cell tumors, sex cord-stromaltumors), Fallopian tubes (e.g., serous adenocarcinoma, mucinousadenocarcinoma, endometrioid adenocarcinoma, clear cell adenocarcinoma,transitional cell carcinoma, squamous cell carcinoma, undifferentiatedcarcinoma, mullerian tumors, adenosarcoma, leiomyosarcoma, teratoma,germ cell tumors, choriocarcinoma, trophoblastic tumors), uterus (e.g.,carcinoma of the cervix, endometrial polyps, endometrial hyperplasia,intraepithelial carcinoma (EIC), endometrial carcinoma (e.g.,endometrioid carcinoma, serous carcinoma, clear cell carcinoma, mucinouscarcinoma, squamous cell carcinoma, transitional carcinoma, small cellcarcinoma, undifferentiated carcinoma, mesenchymal neoplasia), leiomyoma(e.g., endometrial stromal nodule, leiomyosarcoma, endometrial stromalsarcoma (ESS), mesenchymal tumors), mixed epithelial and mesenchymaltumors (e.g., adenofibroma, carcinofibroma, adenosarcoma, carcinosarcoma(malignant mixed mesodermal sarcoma—MMMT)), endometrial stromal tumors,endometrial malignant mullerian mixed tumours, gestational trophoblastictumors (partial hydatiform mole, complete hydatiform mole, invasivehydatiform mole, placental site tumour)), vulva, vagina; male sex organtissues, including prostate, testis (e.g., germ cell tumors,spermatocytic seminoma), penis; bladder (e.g., squamous cell carcinoma,urothelial carcinoma, bladder urothelial carcinoma); brain, (e.g.,gliomas (e.g., astrocytomas, including non-infiltrating, low-grade,anaplastic, glioblastomas; oligodendrogliomas, ependymomas),meningiomas, gangliogliomas, schwannomas (neurilemmomas),craniopharyngiomas, chordomas, Non-Hodgkin lymphomas, pituitary tumors;eye (e.g., retinoma, retinoblastoma, ocular melanoma, posterior uvealmelanoma, iris hamartoma); head and neck (e.g., nasopharyngealcarcinoma, Endolymphatic Sac Tumor (ELST), epidermoid carcinoma,laryngeal cancers including squamous cell carcinoma (SCC) (e.g., glotticcarcinoma, supraglottic carcinoma, subglottic carcinoma, transglotticcarcinoma), carcinoma in situ, verrucous, spindle cell and basaloid SCC,undifferentiated carcinoma, laryngeal adenocarcinoma, adenoid cysticcarcinoma, neuroendocrine carcinomas, laryngeal sarcoma), head and neckparagangliomas (e.g., carotid body, jugulotympanic, vagal); thymus(e.g., thymoma); heart (e.g., cardiac myxoma); lung (e.g., small cellcarcinoma (SCLC), non-small cell lung carcinoma (NSCLC), includingsquamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma,carcinoids (typical or atypical), carcinosarcomas, pulmonary blastomas,giant cell carcinomas, spindle cell carcinomas, pleuropulmonaryblastoma); lymph (e.g., lymphomas, including Hodgkin's lymphoma,non-Hodgkin's lymphoma, Epstein-Barr virus (EBV)-associatedlymphoproliferative diseases, including B cell lymphomas and T celllymphomas (e.g., Burkitt lymphoma, large B cell lymphoma, diffuse largeB-cell lymphoma (DLBCL), mantle cell lymphoma, indolent B-cell lymphoma,low grade B cell lymphoma, fibrin-associated diffuse large celllymphoma; primary effusion lymphoma; plasmablastic lymphoma; extranodalNK/T cell lymphoma, nasal type; peripheral T cell lymphoma, cutaneous Tcell lymphoma, angioimmunoblastic T cell lymphoma; follicular T celllymphoma; systemic T cell lymphoma), lymphangioleiomyomatosis); centralnervous system (CNS) (e.g., gliomas including astrocytic tumors (e.g.,pilocytic astrocytoma, pilomyxoid astrocytoma, subependymal giant cellastrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma,fibrillary astrocytoma, gemistocytic astrocytoma, protoplasmicastrocytoma, anaplastic astrocytoma, glioblastoma (e.g., giant cellglioblastoma, gliosarcoma, glioblastoma multiforme) and gliomatosiscerebri), oligodendroglial tumors (e.g., oligodendroglioma, anaplasticoligodendroglioma), oligoastrocytic tumors (e.g., oligoastrocytoma,anaplastic oligoastrocytoma), ependymal tumors (e.g., subependymom,myxopapillary ependymoma, ependymomas (e.g., cellular, papillary, clearcell, tanycytic), anaplastic ependymoma), optic nerve glioma, andnon-gliomas (e.g., choroid plexus tumors, neuronal and mixedneuronal-glial tumors, pineal region tumors, embryonal tumors,medulloblastoma, meningeal tumors, primary CNS lymphomas, germ celltumors, pituitary adenomas, cranial and paraspinal nerve tumors, stellarregion tumors), neurofibroma, meningioma, peripheral nerve sheathtumors, peripheral neuroblastic tumours (including without limitationneuroblastoma, ganglioneuroblastoma, ganglioneuroma), trisomy 19ependymoma); neuroendocrine tissues (e.g., paraganglionic systemincluding adrenal medulla (pheochromocytomas) and extra-adrenalparaganglia ((extra-adrenal) paragangliomas); skin (e.g., clear cellhidradenoma, cutaneous benign fibrous histiocytomas, cylindroma,hidradenoma, melanoma (including cutaneous melanoma, mucosal melanoma),pilomatricoma, Spitz tumors); and soft tissues (e.g., aggressiveangiomyxoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma,angiofibroma, angiomatoid fibrous histiocytoma, synovial sarcoma,biphasic synovial sarcoma, clear cell sarcoma, dermatofibrosarcomaprotuberans, desmoid-type fibromatosis, small round cell tumor,desmoplastic small round cell tumor, elastofibroma, embryonalrhabdomyosarcoma, Ewing's tumors/primitive neurectodermal tumors (PNET),extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma,paraspinal sarcoma, inflammatory myofibroblastic tumor, lipoblastoma,lipoma, chondroid lipoma, liposarcoma/malignant lipomatous tumors,liposarcoma, myxoid liposarcoma, fibromyxoid sarcoma,lymphangioleiomyoma, malignant myoepithelioma, malignant melanoma ofsoft parts, myoepithelial carcinoma, myoepithelioma, myxoinflammatoryfibroblastic sarcoma, undifferentiated sarcoma, pericytoma,rhabdomyosarcoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), softtissue leiomyosarcoma, undifferentiated sarcoma, well-differentiatedliposarcoma. In certain embodiments, the cancer is selected from thegroup consisting of a melanoma, a gastric cancer, a triple-negativebreast cancer (TNBC), a non-small cell lung cancer (NSCLC), a rectaladenocarcinoma, a colorectal cancer, a renal cell carcinoma, an ovariancancer, a prostate cancer, an oral squamous cell carcinoma (SCC), a headand neck squamous cell carcinoma (HNSCC), a urothelial bladder cancer, aglioblastoma (GBM), a meningioma, adrenal cancer, and an endometrialcancer.

In another aspect, the instant disclosure provides the use of anisolated antibody, polynucleotide, vector, lipid nanoparticle, orpharmaceutical composition as disclosed herein in any of the foregoingmethods for the treatment of cancer.

In another aspect, the instant disclosure provides an isolated antibody,polynucleotide, vector, lipid nanoparticle, or pharmaceuticalcomposition as disclosed herein, for use in any one of the foregoingmethods for the treatment of cancer.

Additional or alternative embodiments of the invention disclosed hereinare set forth below.

-   -   Embodiment 1. An antibody that binds to human CD73, the antibody        comprising a heavy chain variable region comprising        complementarity determining regions (CDRs) CDRH1, CDRH2, and        CDRH3, and a light chain variable region comprising        complementarity determining regions CDRL1, CDRL2, and CDRL3,        wherein:    -   (a) CDRH1 comprises the amino acid sequence of X₁X₂WX₃X₄(SEQ ID        NO: 13), wherein        -   X₁ is S or N;        -   X₂ is S or Y;        -   X₃ is I or M; and        -   X₄ is N or H;    -   (b) CDRH2 comprises the amino acid sequence of        X₁IYPRX₂X₃DTNYX₄X₅KFKX₆ (SEQ ID NO: 14), wherein        -   X₁ is R or T;        -   X₂ is N, A, or S;        -   X₃ is G or S;        -   X₄ is N, A, or S;        -   X₅ is G or Q; and        -   X₆ is D or G;    -   (c) CDRH3 comprises the amino acid sequence of LLDYSMDY (SEQ ID        NO: 7);    -   (d) CDRL1 comprises the amino acid sequence of RASQDISX₁X₂LN        (SEQ ID NO: 16), wherein        -   X₁ is N or I; and        -   X₂ is Y or S;    -   (e) CDRL2 comprises the amino acid sequence of YTSRLHS (SEQ ID        NO: 10); and/or    -   (f) CDRL3 comprises the amino acid sequence of QQGNTLPXT (SEQ ID        NO: 17), wherein: X is L or W.    -   Embodiment 2. The antibody of embodiment 1, wherein CDRH1        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 1 and 2.    -   Embodiment 3. The antibody of embodiment 1 or 2, wherein CDRH2        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 3-6.    -   Embodiment 4. The antibody of any one of the preceding        embodiments, wherein CDRL1 comprises an amino acid sequence        selected from the group consisting of SEQ ID NOs: 8 and 9.    -   Embodiment 5. The antibody of any one of the preceding        embodiments, wherein CDRL3 comprises an amino acid sequence        selected from the group consisting of SEQ ID NOs: 11 and 12.    -   Embodiment 6. The antibody of any one of the preceding        embodiments, wherein:    -   (a) CDRH1 comprises the amino acid sequence of SSWIN (SEQ ID NO:        2);    -   (b) CDRH2 comprises the amino acid sequence of        RIYPRX₁GDTNYX₂GKFKD (SEQ ID NO: 15), wherein        -   X₁ is N, A, or S; and        -   X₂ is N, A, or S;    -   (c) CDRL1 comprises the amino acid sequence of RASQDISX₁X₂LN        (SEQ ID NO: 16), wherein        -   X₁ is N or I; and        -   X₂ is Y or S; and/or    -   (d) CDRL3 comprises the amino acid sequence of QQGNTLPLT (SEQ ID        NO: 12).    -   Embodiment 7. The antibody of embodiment 6, wherein CDRH2        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 4-6.    -   Embodiment 8. The antibody of embodiment 6 or 7, wherein CDRL1        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 8 and 9.    -   Embodiment 9. The antibody of any one of the preceding        embodiments, wherein CDRH1, CDRH2, and CDRH3 comprise the CDRH1,        CDRH2, and CDRH3 amino acid sequences, respectively, set forth        in SEQ ID NOs: 2, 4, and 7; 2, 5, and 7; 1, 3, and 7; or 2, 6,        and 7.    -   Embodiment 10. The antibody of any one of the preceding        embodiments, wherein CDRL1, CDRL2, and CDRL3 comprise the CDRL1,        CDRL2, and CDRL3 amino acid sequences, respectively, set forth        in SEQ ID NOs: 8, 10, and 12; 9, 10, and 12; or 8, 10, and 11.    -   Embodiment 11. The antibody of any one of the preceding        embodiments, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and        CDRL3 comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3        amino acid sequences, respectively, set forth in SEQ ID NOs: 2,        4, 7, 8, 10, and 12; 2, 5, 7, 8, 10, and 12; 1, 3, 7, 8, 10, and        11; 2, 4, 7, 8, 10, and 11; 2, 4, 7, 9, 10, and 12; 2, 6, 7, 8,        10, and 12; 2, 5, 7, 9, 10, and 12; or 2, 6, 7, 9, 10, and 12.    -   Embodiment 12. The antibody of embodiment 11, wherein CDRH1,        CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the CDRH1, CDRH2,        CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences,        respectively, set forth in SEQ ID NOs: 2, 4, 7, 8, 10, and 12;        or 2, 5, 7, 8, 10, and 12.    -   Embodiment 13. An antibody that binds to human CD73, the        antibody comprising a heavy chain variable region comprising        CDRs CDRH1, CDRH2, and CDRH3, and a light chain variable region        comprising complementarity determining regions CDRL1, CDRL2, and        CDRL3, wherein CDRH3 comprises the amino acid sequence of        LLDYSMDY (SEQ ID NO: 7); and/or CDRL3 comprises the amino acid        sequence of QQGNTLPLT (SEQ ID NO: 12) or QQGNTLPWT (SEQ ID NO:        11).    -   Embodiment 14. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a heavy chain        variable region comprising the amino acid sequence of SEQ ID NO:        31.    -   Embodiment 15. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a heavy chain        variable region comprising an amino acid sequence at least 75%,        80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or        100% identical to an amino acid sequence selected from the group        consisting of SEQ ID NOs: 19-21 and 23-30.    -   Embodiment 16. The antibody of embodiment 15, wherein the heavy        chain variable region comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs: 19-21 and 23-30.    -   Embodiment 17. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a heavy chain        variable region comprising an amino acid sequence at least 75%,        80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or        100% identical to an amino acid sequence selected from the group        consisting of SEQ ID NOs: 23-30.    -   Embodiment 18. The antibody of embodiment 17, wherein the heavy        chain variable region comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs: 23-30.    -   Embodiment 19. The antibody of embodiment 18, wherein the heavy        chain variable region comprises the amino acid sequence of SEQ        ID NO: 24 or 27.    -   Embodiment 20. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a heavy chain        variable region comprising an amino acid sequence derived from a        human germline sequence as set forth in SEQ ID NO: 22.    -   Embodiment 21. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a light chain        variable region comprising an amino acid sequence of SEQ ID NO:        41 or 42.    -   Embodiment 22. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a light chain        variable region comprising an amino acid sequence at least 75%,        80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or        100% identical to an amino acid sequence selected from the group        consisting of SEQ ID NOs: 33-35 and 37-40.    -   Embodiment 23. The antibody of embodiment 22, wherein the light        chain variable region comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs: 33-35 and 37-40.    -   Embodiment 24. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a light chain        variable region comprising an amino acid sequence at least 75%,        80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or        100% identical to an amino acid sequence selected from the group        consisting of SEQ ID NOs: 37-40.    -   Embodiment 25. The antibody of embodiment 24, wherein the light        chain variable region comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs: 37-40.    -   Embodiment 26. The antibody of embodiment 25, wherein the light        chain variable region comprises the amino acid sequence of SEQ        ID NO: 37.    -   Embodiment 27. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a light chain        variable region comprising an amino acid sequence derived from a        human germline sequence as set forth in SEQ ID NO: 36.    -   Embodiment 28. The antibody of any one of the preceding        embodiments, wherein the heavy chain variable region and the        light chain variable region, respectively, comprise the amino        acid sequences set forth in SEQ ID NOs: 24 and 37; 27 and 37; 19        and 33; 20 and 34; 21 and 35; 23 and 37; 23 and 38; 24 and 38;        25 and 37; 26 and 37; 28 and 37; 25 and 38; 26 and 38; 27 and        38; 28 and 38; 29 and 37; 30 and 37; 23 and 39; or 23 and 40.    -   Embodiment 29. The antibody of embodiment 28, wherein the heavy        chain variable region and the light chain variable region,        respectively, comprise the amino acid sequences set forth in SEQ        ID NOs: 24 and 37; or 27 and 37.    -   Embodiment 30. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a heavy chain        constant region selected from the group consisting of human        IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂.    -   Embodiment 31. The antibody of embodiment 30, wherein the heavy        chain constant region is IgG₁.    -   Embodiment 32. The antibody of embodiment 31, wherein the amino        acid sequence of IgG₁ comprises a N297A mutation, numbered        according to the EU numbering system.    -   Embodiment 33. The antibody of embodiment 32, wherein the heavy        chain constant region comprises the amino acid sequence of SEQ        ID NO: 50, 49, 46, 45, 55, or 56.    -   Embodiment 34. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a heavy chain        comprising an amino acid sequence selected from the group        consisting of SEQ ID NOs: 63-88.    -   Embodiment 35. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a light chain        constant region selected from the group consisting of human Igκ        and Igλ.    -   Embodiment 36. The antibody of embodiment 35, wherein the light        chain constant region is Igκ.    -   Embodiment 37. The antibody of embodiment 36, wherein the light        chain constant region comprises the amino acid sequence of SEQ        ID NO: 93 or 89.    -   Embodiment 38. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a light chain        comprising the amino acid sequence selected from the group        consisting of SEQ ID NO: 92 and 94-98.    -   Embodiment 39. The antibody of any one of the preceding        embodiments, wherein the antibody comprises a heavy chain and a        light chain comprising the amino acid sequences set forth in any        row of Table 4, preferably the amino acid sequence selected from        the group consisting of SEQ ID NOs: 79 and 97; 85 and 97; 77 and        97; 80 and 97; 86 and 97; 64 and 92; 70 and 96; or 78 and 97,        respectively.    -   Embodiment 40. The antibody of any one of embodiments 15-39,        wherein the X in any one of SEQ ID NOs: 19, 20, and 65-68 is        glutamine.    -   Embodiment 41. The antibody of any one of embodiments 15-39,        wherein the X in any one of SEQ ID NOs: 21, 23-30, 63, 64, 69-88        is glutamate.    -   Embodiment 42. The antibody of any one of embodiments 15-39,        wherein the X in any one of SEQ ID NOs: 19-21, 23-30, and 63-88        is pyroglutamate.    -   Embodiment 43. An antibody that binds to the same epitope of        human CD73 as the antibody of any one of the preceding        embodiments.    -   Embodiment 44. The antibody of any one of the preceding        embodiments, wherein the antibody is a humanized antibody.    -   Embodiment 45. The antibody of any one of the preceding        embodiments, wherein the antibody is antagonistic to human CD73.    -   Embodiment 46. The antibody of embodiment 45, wherein the        antibody deactivates, reduces, or inhibits an activity of human        CD73.    -   Embodiment 47. The antibody of embodiment 45 or 46, wherein the        antibody attenuates the ability of human CD73 to convert        adenosine monophosphate to adenosine.    -   Embodiment 48. The antibody of any one of the preceding        embodiments, wherein the antibody is internalized upon binding        to cells expressing human CD73.    -   Embodiment 49. The antibody of any one of embodiments 1-48,        further comprising a TGFβ-binding moiety.    -   Embodiment 50. The antibody of embodiment 49, wherein the        TGFβ-binding moiety is linked to the heavy chain variable        region.    -   Embodiment 51. The antibody of embodiment 50, wherein the        TGFβ-binding moiety is linked to the heavy chain variable region        via a peptide linker.    -   Embodiment 52. The antibody of any one of embodiments 30-48,        further comprising a TGFβ-binding moiety linked to the heavy        chain constant region.    -   Embodiment 53. The antibody of embodiment 52, wherein the        TGFβ-binding moiety is linked to the C-terminal residue of the        heavy chain constant region.    -   Embodiment 54. The antibody of embodiment 52 or 53, wherein the        N-terminal residue of the TGFβ-binding moiety is linked to the        C-terminal residue of the heavy chain constant region via a        peptide linker.    -   Embodiment 55. The antibody of embodiment 51 or 54, wherein the        peptide linker comprises an amino acid sequence selected from        the group consisting of SEQ ID NOs: 103-108.    -   Embodiment 56. The antibody of any one of embodiments 49-55,        wherein the TGFβ-binding moiety binds to human TGFβ.    -   Embodiment 57. The antibody of any one of embodiments 49-56,        wherein the TGFβ-binding moiety comprises an extracellular        domain of a human TGFβ receptor.    -   Embodiment 58. The antibody of embodiment 57, wherein the human        TGFβ receptor is selected from the group consisting of human        TGFβR1, TGFβR2, and TGFβR3.    -   Embodiment 59. The antibody of embodiment 57 or 58, wherein the        human TGFβ receptor is human TGFβR2.    -   Embodiment 60. The antibody of any one of embodiments 49-59,        wherein the TGFβ-binding moiety comprises an amino acid sequence        at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to a sequence selected from the group        consisting of SEQ ID NOs: 109-112.    -   Embodiment 61. The antibody of any one of embodiments 49-60,        wherein the TGFβ-binding moiety consists of an amino acid        sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, or 99% identical to a sequence selected from the        group consisting of SEQ ID NOs: 109-112.    -   Embodiment 62. The antibody of any one of embodiments 49-61,        wherein the TGFβ-binding moiety comprises an amino acid sequence        selected from the group consisting of SEQ ID NOs: 109-112,        preferably SEQ ID NO: 111.    -   Embodiment 63. The antibody of any one of embodiments 49-62,        wherein the TGFβ-binding moiety consists of an amino acid        sequence selected from the group consisting of SEQ ID NOs:        109-112, preferably SEQ ID NO: 111.    -   Embodiment 64. The antibody of any one of embodiments 49-63, the        antibody comprising a polypeptide comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:        113-121, preferably SEQ ID NO: 114 or 115.    -   Embodiment 65. The antibody of any one of embodiments 49-64,        wherein the antibody comprises a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NOs:        113-121 and a polypeptide comprising the amino acid sequence of        SEQ ID NO: 92 or 97.    -   Embodiment 66. The antibody of any one of embodiments 49-65,        wherein the antibody comprises two polypeptides each comprising        an amino acid sequence selected from the group consisting of SEQ        ID NOs: 113-121 and two polypeptides each comprising the amino        acid sequence of SEQ ID NO: 92 or 97.    -   Embodiment 67. The antibody of any one of embodiments 49-66,        wherein the antibody comprises two polypeptides each comprising        the amino acid sequence of SEQ ID NO: 115 and two polypeptides        each comprising the amino acid sequence of SEQ ID NO: 97.    -   Embodiment 68. An antibody that binds to human CD73, the        antibody comprising a TGFβ-binding moiety.    -   Embodiment 69. The antibody of embodiment 68, wherein the        antibody is antagonistic to human CD73.    -   Embodiment 70. The antibody of embodiment 69, wherein the        antibody deactivates, reduces, or inhibits an activity of human        CD73.    -   Embodiment 71. The antibody of embodiment 69 or 70, wherein the        antibody attenuates the ability of human CD73 to convert        adenosine monophosphate to adenosine.    -   Embodiment 72. The antibody of any one of embodiments 68-71,        wherein the antibody, when bound to TGFβ, is antagonistic to        human CD73.    -   Embodiment 73. The antibody of any one of embodiments 68-72,        wherein the TGFβ-binding moiety binds to human TGFβ.    -   Embodiment 74. The antibody of any one of embodiments 68-73,        wherein the TGFβ-binding moiety comprises an extracellular        domain of a human TGFβ receptor.    -   Embodiment 75. The antibody of any one of embodiments 68-74,        wherein the human TGFβ receptor is selected from the group        consisting of human TGFβR1, TGFβR2, and TGFβR3.    -   Embodiment 76. The antibody of embodiment 74 or 75, wherein the        human TGFβ receptor is human TGFβR2.    -   Embodiment 77. The antibody of any one of embodiments 68-76,        wherein the TGFβ-binding moiety comprises an amino acid sequence        at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to a sequence selected from the group        consisting of SEQ ID NOs: 109-112.    -   Embodiment 78. The antibody of any one of embodiments 68-77,        wherein the TGFβ-binding moiety consists of an amino acid        sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, or 99% identical to a sequence selected from the        group consisting of SEQ ID NOs: 109-112.    -   Embodiment 79. The antibody of any one of embodiments 68-78,        wherein the TGFβ-binding moiety comprises an amino acid sequence        selected from the group consisting of SEQ ID NOs: 109-112,        preferably SEQ ID NO: 111.    -   Embodiment 80. The antibody of any one of embodiments 68-79,        wherein the TGFβ-binding moiety consists of an amino acid        sequence selected from the group consisting of SEQ ID NOs:        109-112, preferably SEQ ID NO: 111.    -   Embodiment 81. The antibody of any one of embodiments 1-48,        further comprising a VEGF-binding moiety.    -   Embodiment 82. The antibody of embodiment 81, wherein the        VEGF-binding moiety is linked to the heavy chain variable        region.    -   Embodiment 83. The antibody of embodiment 82, wherein the        VEGF-binding moiety is linked to the heavy chain variable region        via a peptide linker.    -   Embodiment 84. The antibody of any one of embodiments 30-48,        further comprising a VEGF-binding moiety linked to the heavy        chain constant region.    -   Embodiment 85. The antibody of embodiment 84, wherein the        VEGF-binding moiety is linked to the C-terminal residue of the        heavy chain constant region.    -   Embodiment 86. The antibody of embodiment 84 or 85, wherein the        N-terminal residue of the VEGF-binding moiety is linked to the        C-terminal residue of the heavy chain constant region via a        peptide linker.    -   Embodiment 87. The antibody of embodiment 83 or 86, wherein the        peptide linker comprises an amino acid sequence selected from        the group consisting of SEQ ID NOs: 103-108.    -   Embodiment 88. The antibody of any one of embodiments 81-87,        wherein the VEGF-binding moiety binds to human VEGF.    -   Embodiment 89. The antibody of any one of embodiments 81-88,        wherein the VEGF-binding moiety comprises an extracellular        domain of a human VEGF receptor.    -   Embodiment 90. The antibody of any one of embodiments 81-89,        wherein the human VEGF receptor is selected from the group        consisting of human VEGFR1, VEGFR2, and VEGFR3.    -   Embodiment 91. The antibody of embodiment 89 or 90, wherein the        human VEGF receptor is human VEGFR1.    -   Embodiment 92. The antibody of any one of embodiments 81-91,        wherein the VEGF-binding moiety comprises an amino acid sequence        at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to the sequence set forth in SEQ ID NO:        122.    -   Embodiment 93. The antibody of any one of embodiments 81-92,        wherein the VEGF-binding moiety consists of an amino acid        sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ        ID NO: 122.    -   Embodiment 94. The antibody of any one of embodiments 81-93,        wherein the VEGF-binding moiety comprises the sequence set forth        in SEQ ID NO: 122.    -   Embodiment 95. The antibody of any one of embodiments 81-94,        wherein the VEGF-binding moiety consists of the sequence set        forth in SEQ ID NO: 122.    -   Embodiment 96. The antibody of any one of embodiments 81-92, the        antibody comprising a polypeptide comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs:        123-127.    -   Embodiment 97. The antibody of any one of embodiments 81-96,        wherein the antibody comprises a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NOs:        123-127 and a polypeptide comprising an amino acid sequence        selected from the group consisting of SEQ ID NOs: 92, 128, and        97.    -   Embodiment 98. The antibody of any one of embodiments 81-97,        wherein the antibody comprises two polypeptides comprising an        amino acid sequence selected from the group consisting of SEQ ID        NOs: 123-127 and two polypeptides comprising an amino acid        sequence selected from the group consisting of SEQ ID NOs: 92,        128, and 97.    -   Embodiment 99. An antibody that binds to human CD73, the        antibody comprising a VEGF-binding moiety.    -   Embodiment 100. The antibody of embodiment 99, wherein the        antibody is antagonistic to human CD73.    -   Embodiment 101. The antibody of embodiment 100, wherein the        antibody deactivates, reduces, or inhibits an activity of human        CD73.    -   Embodiment 102. The antibody of embodiment 100, wherein the        antibody attenuates the ability of human CD73 to convert        adenosine monophosphate to adenosine.    -   Embodiment 103. The antibody of any one of embodiments 99-102,        wherein the antibody, when bound to VEGF, is antagonistic to        human CD73.    -   Embodiment 104. The antibody of embodiment any one of        embodiments 99-103, wherein the VEGF-binding moiety binds to        human VEGF.    -   Embodiment 105. The antibody of any one of embodiments 99-104,        wherein the VEGF-binding moiety comprises an extracellular        domain of a human VEGF receptor.    -   Embodiment 106. The antibody of any one of embodiments 99-105,        wherein the human VEGF receptor is selected from the group        consisting of human VEGFR1, VEGFR2, and VEGFR3.    -   Embodiment 107. The antibody of embodiment 105 or 106, wherein        the human VEGF receptor is human VEGFR1.    -   Embodiment 108. The antibody of any one of embodiments 99-107,        wherein the VEGF-binding moiety comprises an amino acid sequence        at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to the sequence set forth in SEQ ID NO:        122.    -   Embodiment 109. The antibody of any one of embodiments 99-108,        wherein the VEGF-binding moiety consists of an amino acid        sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ        ID NO: 122.    -   Embodiment 110. The antibody of any one of embodiments 99-109,        wherein the VEGF-binding moiety comprises the amino acid        sequence as set forth in SEQ ID NO: 122.    -   Embodiment 111. The antibody of any one of embodiments 99-110,        wherein the VEGF-binding moiety consists of the amino acid        sequence as set forth in SEQ ID NO: 122.    -   Embodiment 112. The antibody of any one of the preceding        embodiments, further comprising a conjugated cytotoxic agent,        cytostatic agent, toxin, radionuclide, or detectable label.    -   Embodiment 113. The antibody of any one of the preceding        embodiments, wherein the antibody binds to an epitope located        within a region of human CD73, the amino acid sequence of the        region consisting of the amino acid sequence of SEQ ID NOs: 90        and/or 91.    -   Embodiment 114. The antibody of any one of the preceding        embodiments, wherein the antibody binds to at least one residue        in the amino acid sequence of SEQ ID NOs: 90 and/or 91.    -   Embodiment 115. The antibody of any one of the preceding        embodiments, wherein the antibody binds to one or more amino        acid residues of human CD73 selected from the group consisting        of Y158, Y161, P165, or D168, numbered according to the amino        acid sequence of SEQ ID NO: 99.    -   Embodiment 116. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 133 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 117. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 134 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 118. The antibody of any one of the preceding        embodiments, wherein the antibody binds to amino acid residue        Y158 of human CD73, numbered according to the amino acid        sequence of SEQ ID NO: 99.    -   Embodiment 119. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 59 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 120. The antibody of any one of the preceding        embodiments, wherein the antibody binds to amino acid residue        Y161 of human CD73, numbered according to the amino acid        sequence of SEQ ID NO: 99.    -   Embodiment 121. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 60 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 122. The antibody of any one of the preceding        embodiments, wherein the antibody binds to amino acid residue        P165 of human CD73, numbered according to the amino acid        sequence of SEQ ID NO: 99.    -   Embodiment 123. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 139 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 124. The antibody of any one of the preceding        embodiments, wherein the antibody binds to amino acid residue        D168 of human CD73, numbered according to the amino acid        sequence of SEQ ID NO: 99.    -   Embodiment 125. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 140 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 126. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 135 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 127. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 136 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 128. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 142 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141, and wherein the binding affinity of the antibody for        a protein comprising the amino acid sequence of SEQ ID NO: 137        is not substantially reduced relative to the binding affinity of        the antibody for a protein comprising the amino acid sequence of        SEQ ID NO: 141.    -   Embodiment 129. The antibody of any one of the preceding        embodiments, wherein the binding affinity of the antibody to a        protein comprising the amino acid sequence of SEQ ID NO: 142 is        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141, and wherein the binding affinity of the antibody for        a protein comprising the amino acid sequence of SEQ ID NO: 138        is not substantially reduced relative to the binding affinity of        the antibody for a protein comprising the amino acid sequence of        SEQ ID NO: 141.    -   Embodiment 130. An antibody that binds to human CD73, wherein        the antibody binds to an epitope located within a region of        human CD73, the amino acid sequence of the region consisting of        the amino acid sequence of SEQ ID NOs: 90 and/or 91.    -   Embodiment 131. An antibody that binds to human CD73, wherein        the antibody binds to at least one residue in the amino acid        sequence of SEQ ID NOs: 90 and/or 91.    -   Embodiment 132. An antibody that binds to human CD73, wherein        the antibody binds to one or more amino acid residues of human        CD73 selected from the group consisting of Y158, Y161, P165, or        D168, numbered according to the amino acid sequence of SEQ ID        NO: 99.    -   Embodiment 133. An antibody that binds to human CD73, wherein        the binding affinity of the antibody for a protein comprising        the amino acid sequence of SEQ ID NO: 133 is substantially        reduced relative to the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 141.    -   Embodiment 134. An antibody that binds to human CD73, wherein        the binding affinity of the antibody for a protein comprising        the amino acid sequence of SEQ ID NO: 134 is substantially        reduced relative to the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 141.    -   Embodiment 135. An antibody that binds to human CD73, wherein        the antibody binds to amino acid residue Y158 of human CD73,        numbered according to the amino acid sequence of SEQ ID NO: 99.    -   Embodiment 136. An antibody that binds to human CD73, wherein        the binding affinity of the antibody for a protein comprising        the amino acid sequence of SEQ ID NO: 59 is substantially        reduced relative to the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 141.    -   Embodiment 137. An antibody that binds to human CD73, wherein        the antibody binds to amino acid residue Y161 of human CD73,        numbered according to the amino acid sequence of SEQ ID NO: 99.    -   Embodiment 138. An antibody that binds to human CD73, wherein        the binding affinity of the antibody for a protein comprising        the amino acid sequence of SEQ ID NO: 60 is substantially        reduced relative to the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 141.    -   Embodiment 139. An antibody that binds to human CD73, wherein        the antibody binds to amino acid residue P165 of human CD73,        numbered according to the amino acid sequence of SEQ ID NO: 99.    -   Embodiment 140. An antibody that binds to human CD73, wherein        the binding affinity of the antibody for a protein comprising        the amino acid sequence of SEQ ID NO: 139 is substantially        reduced relative to the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 141.    -   Embodiment 141. An antibody that binds to human CD73, wherein        the antibody binds to amino acid residue D168 of human CD73,        numbered according to the amino acid sequence of SEQ ID NO: 99.    -   Embodiment 142. An antibody that binds to human CD73, wherein        the binding affinity of the antibody for a protein comprising        the amino acid sequence of SEQ ID NO: 140 is substantially        reduced relative to the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 141.    -   Embodiment 143. An antibody that binds to human CD73, wherein        the binding affinity of the antibody for a protein comprising        the amino acid sequence of SEQ ID NO: 135 is substantially        reduced relative to the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 141.    -   Embodiment 144. An antibody that binds to human CD73, wherein        the binding affinity of the antibody for a protein comprising        the amino acid sequence of SEQ ID NO: 136 is substantially        reduced relative to the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 141.    -   Embodiment 145. An antibody that binds to human CD73, wherein        the binding affinity of the antibody for a protein comprising        the amino acid sequence of SEQ ID NO: 142 is substantially        reduced relative to the binding affinity of the antibody for a        protein comprising the amino acid sequence of SEQ ID NO: 141,        and wherein the binding affinity of the antibody for a protein        comprising the amino acid sequence of SEQ ID NO: 137 is not        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 146. An antibody that binds to human CD73, wherein        the binding affinity of the antibody to a protein comprising the        amino acid sequence of SEQ ID NO: 142 is substantially reduced        relative to the binding affinity of the antibody for a protein        comprising the amino acid sequence of SEQ ID NO: 141, and        wherein the binding affinity of the antibody for a protein        comprising the amino acid sequence of SEQ ID NO: 138 is not        substantially reduced relative to the binding affinity of the        antibody for a protein comprising the amino acid sequence of SEQ        ID NO: 141.    -   Embodiment 147. A compound or molecule comprising a CD73-binding        moiety and a TGFβ-binding moiety.    -   Embodiment 148. A polynucleotide encoding a polypeptide of the        antibody of any one of embodiments 1-146.    -   Embodiment 149. The polynucleotide of embodiment 148, wherein        the polynucleotide comprises cDNA or mRNA.    -   Embodiment 150. A vector comprising the polynucleotide of        embodiment 148 or 149.    -   Embodiment 151. The vector of embodiment 150, wherein the vector        is a plasmid vector or a viral vector.    -   Embodiment 152. A recombinant host cell comprising the        polynucleotide of embodiment 148 or 149, or the vector of        embodiment 150 or 151.    -   Embodiment 153. A lipid nanoparticle comprising the        polynucleotide of embodiment 148 or 149, or the vector of        embodiment 150 or 151.    -   Embodiment 154. A pharmaceutical composition comprising the        antibody, polynucleotide, vector, or lipid nanoparticle of any        one of embodiments 1-146 or 148-153 and a pharmaceutically        acceptable carrier or excipient.    -   Embodiment 155. A method of producing an antibody, the method        comprising culturing the host cell of embodiment 152 under        suitable conditions so that the polynucleotide is expressed and        the antibody is produced.    -   Embodiment 156. A method of treating cancer in a subject, the        method comprising administering to the subject an effective        amount of the antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition of any one of        embodiments 1-146, 148-151, 153, or 154.    -   Embodiment 157. A method of inhibiting cancer metastasis in a        subject, the method comprising administering to the subject an        effective amount of the antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition of any one of        embodiments 1-146, 148-151, 153, or 154.    -   Embodiment 158. The method of embodiment 156 or 157, wherein the        antibody, polynucleotide, vector, lipid nanoparticle, or        pharmaceutical composition is administered intravenously,        intratumorally, subcutaneously, intradermally, intramuscularly,        intravesically, intracranially, intracavitary or        intraventricularly.    -   Embodiment 159. The method of embodiment 156 or 157, wherein the        antibody, polynucleotide, vector, lipid nanoparticle, or        pharmaceutical composition is administered intratumorally.    -   Embodiment 160. The method of any one of embodiments 156-159,        further comprising administering one or more additional        therapeutic agents to the subject.    -   Embodiment 161. The method of any one of embodiments 156-160,        wherein the antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition, and the one or more        additional therapeutic agents are co-administered according to        the same schedule (e.g., co-administered at the same time        intervals).    -   Embodiment 162. The method of any one of embodiments 156-160,        wherein the antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition, and the one or more        additional therapeutic agents are co-administered according to        different schedules (e.g., co-administered at different time        intervals).    -   Embodiment 163. The method of any one of embodiments 156-162,        wherein the antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition is co-administered        with one or more additional therapeutic agents comprising an        activator or agonist of a fms related tyrosine kinase 3 (FLT3;        CD135) receptor, a toll-like receptor (TLR) or a stimulator of        interferon genes (STING) receptor.    -   Embodiment 164. The method of embodiment 163, wherein the TLR        agonist or activator is selected from the group consisting of a        TLR2 agonist, a TLR3 agonist, a TLR7 agonist, a TLR8 agonist and        a TLR9 agonist.    -   Embodiment 165. The method of embodiment 163, wherein the STING        receptor agonist or activator is selected from the group        consisting of ADU-S100 (MIW-815), SB-11285, MK-1454, SR-8291,        AdVCA0848, GSK-532, SYN-STING, MSA-1, SR-8291,        5,6-dimethylxanthenone-4-acetic acid (DMXAA), cyclic-GAMP        (cGAMP) and cyclic-di-AMP.    -   Embodiment 166. The method of any one of embodiments 156-165,        wherein the antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition is co-administered        with one or more additional therapeutic agents comprising an        inhibitor or antagonist of: protein tyrosine phosphatase,        non-receptor type 11 (PTPN11 or SHP2), myeloid cell leukemia        sequence 1 (MCL1) apoptosis regulator; mitogen-activated protein        kinase kinase kinase kinase 1 (MAP4K1) (also called        Hematopoietic Progenitor Kinase 1 (HPK1)); diacylglycerol kinase        alpha (DGKA, DAGK, DAGK1 or DGK-alpha); 5′-nucleotidase ecto        (NT5E or CD73); transforming growth factor beta 1 (TGFB1 or        TGFβ); heme oxygenase 1 (HMOX1, HO-1 or HO1); vascular        endothelial growth factor A (VEGFA or VEGF); erb-b2 receptor        tyrosine kinase 2 (ERBB2 HER2, HER2/neu or CD340); epidermal        growth factor receptor (EGFR, ERBB, ERBB1 or HER1); ALK receptor        tyrosine kinase (ALK, CD246); poly(ADP-ribose) polymerase 1        (PARP1 or PARP); cyclin dependent kinase 4 (CDK4); cyclin        dependent kinase 6 (CDK6); C—C motif chemokine receptor 8 (CCR8,        CDw198); CD274 molecule (CD274, PDL1 or PD-L1); programmed cell        death 1 (PDCD1, PD1 or PD-1); and/or cytotoxic T-lymphocyte        associated protein 4 (CTLA4, CTLA-4, CD152).    -   Embodiment 167. The method of embodiment 166, wherein the        inhibitor comprises an antigen binding molecule, an antibody or        an antigen-binding fragment thereof.    -   Embodiment 168. The method of embodiment 166, wherein the        inhibitor comprises a small organic molecule.    -   Embodiment 169. The method of embodiment 166, wherein the        inhibitor of MCL1 is selected from the group consisting of        AMG-176, AMG-397, 5-64315, AZD-5991, 483-LM, A 1210477, UMI-77,        and JKY-5-037.    -   Embodiment 170. The method of embodiment 166, wherein the        inhibitor of PTPN11 or SHP2 is selected from the group        consisting of TNO155 (SHP-099), RMC-4550, JAB-3068 and RMC-4630.    -   Embodiment 171. The method of embodiment 160, wherein the        additional therapeutic agent is a chemotherapeutic, an        anti-neoplastic, a radiotherapeutic, or a checkpoint targeting        agent.    -   Embodiment 172. The method of embodiment 160, wherein the one or        more anti-neoplastic or chemotherapeutic agents are selected        from the group consisting of a nucleoside analog (e.g.,        5-fluorouracil, gemcitabine, cytarabine), a taxane (e.g.,        paclitaxel, nab-paclitaxel, docetaxel, cabazitaxel), a platinum        coordination complex (cisplatin, carboplatin, oxaliplatin,        nedaplatin, triplatin tetranitrate, phenanthriplatin,        picoplatin, satraplatin, dicycloplatin, eptaplatin, lobaplatin,        miriplatin), a dihydrofolate reductase (DHFR) inhibitor (e.g.,        methotrexate, trimetrexate, pemetrexed), a topoisomerase        inhibitor (e.g., doxorubicin, daunorubicin, dactinomycin,        eniposide, epirubicin, etoposide, idarubicin, irinotecan,        mitoxantrone, pixantrone, sobuzoxane, topotecan, irinotecan,        MM-398 (liposomal irinotecan), vosaroxin and GPX-150,        aldoxorubicin, AR-67, mavelertinib, AST-2818, avitinib        (ACEA-0010), irofulven (MGI-114)), an alkylating agent (e.g., a        nitrogen mustard (e.g., cyclophosphamide, chlormethine,        uramustine or uracil mustard, melphalan, chlorambucil,        ifosfamide, bendamustine, temozolomide, carmustine), a        nitrosourea (e.g., carmustine, lomustine, streptozocin), an        alkyl sulfonate (e.g., busulfan)), and mixtures thereof.    -   Embodiment 173. The method of embodiment 160, wherein the        checkpoint targeting agent is selected from the group consisting        of an antagonist anti-PD-1 antibody, an antagonist anti-PD-L1        antibody, an antagonist anti-PD-L2 antibody, an antagonist        anti-CTLA-4 antibody, an antagonist anti-TIM-3 antibody, an        antagonist anti-LAG-3 antibody, an antagonist anti-CEACAM1        antibody, an agonist anti-GITR antibody, an antagonist        anti-TIGIT antibody, an antagonist anti-VISTA antibody, an        agonist anti-CD137 antibody, and an agonist anti-OX40 antibody.    -   Embodiment 174. The method of embodiment 173, wherein the        additional therapeutic agent is an anti-PD-1 antibody,        optionally wherein the anti-PD-1 antibody is pembrolizumab or        nivolumab.    -   Embodiment 175. The method of embodiment 160, wherein the        additional therapeutic agent is an inhibitor of        indoleamine-2,3-dioxygenase (IDO).    -   Embodiment 176. The method of embodiment 175, wherein the        inhibitor is selected from the group consisting of epacadostat,        F001287, indoximod, and NLG919.    -   Embodiment 177. The method of embodiment 176, wherein the        inhibitor is epacadostat.    -   Embodiment 178. The method of embodiment 160, wherein the        additional therapeutic agent is a vaccine.    -   Embodiment 179. The method of embodiment 178, wherein the        vaccine comprises a heat shock protein peptide complex (HSPPC)        comprising a heat shock protein complexed with an antigenic        peptide.    -   Embodiment 180. The method of embodiment 179, wherein the heat        shock protein is hsc70 and is complexed with a tumor-associated        antigenic peptide.    -   Embodiment 181. The method of embodiment 179, wherein the heat        shock protein is gp96 and is complexed with a tumor-associated        antigenic peptide, wherein the HSPPC is derived from a tumor        obtained from a subject.    -   Embodiment 182. The method of embodiment 160, wherein the        additional therapeutic agent is etoposide or doxorubicin.    -   Embodiment 183. The method of any one of embodiments 156-182,        wherein the antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition is co-administered        with a FOLFOX regimen, a FOLFOXIRI regimen or a FOLFIRINOX        regimen.    -   Embodiment 184. The method of any one of embodiments 156-183,        wherein the antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition is co-administered        with an immunotherapy, an immunostimulatory therapy, a cellular        therapy or a gene therapy.    -   Embodiment 185. The method of any one of embodiments 156-184,        wherein the cancer is selected from the group consisting of a        solid tumor, a hematological cancer, and a metastatic lesion.    -   Embodiment 186. The method of embodiment 185, wherein the solid        tumor is selected from the group consisting of a sarcoma, a        fibroblastic sarcoma, a carcinoma, and an adenocarcinoma.    -   Embodiment 187. The method of embodiment 185, wherein the        hematological cancer is selected from the group consisting of a        leukemia, a lymphoma, and a myeloma.    -   Embodiment 188. The method of any one of embodiments 156-184,        wherein the cancer is selected from the group consisting of a        lung cancer, a melanoma, a renal cancer, a liver cancer, a        myeloma, a prostate cancer, a breast cancer, an ovarian cancer,        a colorectal cancer, a pancreatic cancer, a head and neck        cancer, an anal cancer, a gastro-esophageal cancer, a        mesothelioma, a nasopharyngeal cancer, a thyroid cancer, a        cervical cancer, an epithelial cancer, a peritoneal cancer, a        lymphoproliferative disease, an acute lymphoblastic leukemia        (ALL), an acute myelogenous leukemia (AML), a chronic        lymphocytic leukemia (CLL), a chronic myelogenous leukemia        (CML), a chronic myelomonocytic leukemia (CMML), a hairy cell        leukemia, a B cell lymphoma, a diffuse large B-cell lymphoma        (DLBCL), an activated B-cell like (ABC) diffuse large B cell        lymphoma, a germinal center B cell (GCB) diffuse large B cell        lymphoma, a mantle cell lymphoma, a Hodgkin lymphoma, a        non-Hodgkin lymphoma, a relapsed non-Hodgkin lymphoma, a        refractory non-Hodgkin lymphoma, a recurrent follicular        non-Hodgkin lymphoma, a Burkitt lymphoma, a small lymphocytic        lymphoma, a follicular lymphoma, a lymphoplasmacytic lymphoma,        and an extranodal marginal zone lymphoma.    -   Embodiment 189. The method of any one of embodiments 156 to 184,        wherein the cancer is selected from the group consisting of an        epithelial tumor (e.g., a carcinoma, a squamous cell carcinoma,        a basal cell carcinoma, a squamous intraepithelial neoplasia), a        glandular tumor (e.g., an adenocarcinoma, an adenoma, an        adenomyoma), a mesenchymal or soft tissue tumor (e.g., a        sarcoma, a rhabdomyosarcoma, a leiomyosarcoma, a liposarcoma, a        fibrosarcoma, a dermatofibrosarcoma, a neurofibrosarcoma, a        fibrous histiocytoma, an angiosarcoma, an angiomyxoma, a        leiomyoma, a chondroma, a chondrosarcoma, an alveolar soft-part        sarcoma, an epithelioid hemangioendothelioma, a Spitz tumor, a        synovial sarcoma), and a lymphoma.    -   Embodiment 190. The method of any one of embodiments 156 to 184,        wherein the cancer is a solid tumor in or arising from a tissue        or organ selected from the group consisting of: bone (e.g.,        adamantinoma, aneurysmal bone cysts, angiosarcoma,        chondroblastoma, chondroma, chondromyxoid fibroma,        chondrosarcoma, chordoma, dedifferentiated chondrosarcoma,        enchondroma, epithelioid hemangioendothelioma, fibrous dysplasia        of the bone, giant cell tumour of bone, haemangiomas and related        lesions, osteoblastoma, osteochondroma, osteosarcoma, osteoid        osteoma, osteoma, periosteal chondroma, Desmoid tumor, Ewing        sarcoma); lips and oral cavity (e.g., odontogenic ameloblastoma,        oral leukoplakia, oral squamous cell carcinoma, primary oral        mucosal melanoma); salivary glands (e.g., pleomorphic salivary        gland adenoma, salivary gland adenoid cystic carcinoma, salivary        gland mucoepidermoid carcinoma, salivary gland Warthin's        tumors); esophagus (e.g., Barrett's esophagus, dysplasia and        adenocarcinoma); gastrointestinal tract, including stomach        (e.g., gastric adenocarcinoma, primary gastric lymphoma,        gastrointestinal stromal tumors (GISTs), metastatic deposits,        gastric carcinoids, gastric sarcomas, neuroendocrine carcinoma,        gastric primary squamous cell carcinoma, gastric        adenoacanthomas), intestines and smooth muscle (e.g.,        intravenous leiomyomatosis), colon (e.g., colorectal        adenocarcinoma), rectum, anus; pancreas (e.g., serous neoplasms,        including microcystic or macrocystic serous cystadenoma, solid        serous cystadenoma, Von Hippel-Landau (VHL)-associated serous        cystic neoplasm, serous cystadenocarcinoma, mucinous cystic        neoplasms (MCN), intraductal papillary mucinous neoplasms        (IPMN), intraductal oncocytic papillary neoplasms (IOPN),        intraductal tubular neoplasms, cystic acinar neoplasms,        including acinar cell cystadenoma, acinar cell        cystadenocarcinoma, pancreatic adenocarcinoma, invasive        pancreatic ductal adenocarcinomas, including tubular        adenocarcinoma, adenosquamous carcinoma, colloid carcinoma,        medullary carcinoma, hepatoid carcinoma, signet ring cell        carcinoma, undifferentiated carcinoma, undifferentiated        carcinoma with osteoclast-like giant cells, acinar cell        carcinoma, neuroendocrine neoplasms, neuroendocrine        microadenoma, neuroendocrine tumors (NET), neuroendocrine        carcinoma (NEC), including small cell or large cell NEC,        insulinoma, gastrinoma, glucagonoma, serotonin-producing NET,        somatostatinoma, VIPoma, solid-pseudopapillary neoplasms (SPN),        pancreatoblastoma); gall bladder (e.g. carcinoma of the        gallbladder and extrahepatic bile ducts, intrahepatic        cholangiocarcinoma); neuro-endocrine (e.g., adrenal cortical        carcinoma, carcinoid tumors, phaeochromocytoma, pituitary        adenomas); thyroid (e.g., anaplastic (undifferentiated)        carcinoma, medullary carcinoma, oncocytic tumors, papillary        carcinoma, adenocarcinoma); liver (e.g., adenoma, combined        hepatocellular and cholangiocarcinoma, fibrolamellar carcinoma,        hepatoblastoma, hepatocellular carcinoma, mesenchymal, nested        stromal epithelial tumor, undifferentiated carcinoma,        hepatocellular carcinoma, intrahepatic cholangiocarcinoma, bile        duct cystadenocarcinoma, epithelioid hemangioendothelioma,        angiosarcoma, embryonal sarcoma, rhabdomyosarcoma, solitary        fibrous tumor, teratoma, York sac tumor, carcinosarcoma,        rhabdoid tumor); kidney (e.g., ALK-rearranged renal cell        carcinoma, chromophobe renal cell carcinoma, clear cell renal        cell carcinoma, clear cell sarcoma, metanephric adenoma,        metanephric adenofibroma, mucinous tubular and spindle cell        carcinoma, nephroma, nephroblastoma (Wilms tumor), papillary        adenoma, papillary renal cell carcinoma, renal oncocytoma, renal        cell carcinoma, succinate dehydrogenase-deficient renal cell        carcinoma, collecting duct carcinoma); breast (e.g., invasive        ductal carcinoma, including without limitation, acinic cell        carcinoma, adenoid cystic carcinoma, apocrine carcinoma,        cribriform carcinoma, glycogen-rich/clear cell, inflammatory        carcinoma, lipid-rich carcinoma, medullary carcinoma,        metaplastic carcinoma, micropapillary carcinoma, mucinous        carcinoma, neuroendocrine carcinoma, oncocytic carcinoma,        papillary carcinoma, sebaceous carcinoma, secretory breast        carcinoma, tubular carcinoma, lobular carcinoma, including        without limitation, pleomorphic carcinoma, signet ring cell        carcinoma, peritoneum (e.g., mesothelioma, primary peritoneal        cancer)); female sex organ tissues, including ovary (e.g.,        choriocarcinoma, epithelial tumors, germ cell tumors, sex        cord-stromal tumors), Fallopian tubes (e.g., serous        adenocarcinoma, mucinous adenocarcinoma, endometrioid        adenocarcinoma, clear cell adenocarcinoma, transitional cell        carcinoma, squamous cell carcinoma, undifferentiated carcinoma,        mullerian tumors, adenosarcoma, leiomyosarcoma, teratoma, germ        cell tumors, choriocarcinoma, trophoblastic tumors), uterus        (e.g., carcinoma of the cervix, endometrial polyps, endometrial        hyperplasia, intraepithelial carcinoma (EIC), endometrial        carcinoma (e.g., endometrioid carcinoma, serous carcinoma, clear        cell carcinoma, mucinous carcinoma, squamous cell carcinoma,        transitional carcinoma, small cell carcinoma, undifferentiated        carcinoma, mesenchymal neoplasia), leiomyoma (e.g., endometrial        stromal nodule, leiomyosarcoma, endometrial stromal sarcoma        (ESS), mesenchymal tumors), mixed epithelial and mesenchymal        tumors (e.g., adenofibroma, carcinofibroma, adenosarcoma,        carcinosarcoma (malignant mixed mesodermal sarcoma—MMMT)),        endometrial stromal tumors, endometrial malignant mullerian        mixed tumours, gestational trophoblastic tumors (partial        hydatiform mole, complete hydatiform mole, invasive hydatiform        mole, placental site tumour)), vulva, vagina; male sex organ        tissues, including prostate, testis (e.g., germ cell tumors,        spermatocytic seminoma), penis; bladder (e.g., squamous cell        carcinoma, urothelial carcinoma, bladder urothelial carcinoma);        brain, (e.g., gliomas (e.g., astrocytomas, including        non-infiltrating, low-grade, anaplastic, glioblastomas;        oligodendrogliomas, ependymomas), meningiomas, gangliogliomas,        schwannomas (neurilemmomas), craniopharyngiomas, chordomas,        Non-Hodgkin lymphomas, pituitary tumors; eye (e.g., retinoma,        retinoblastoma, ocular melanoma, posterior uveal melanoma, iris        hamartoma); head and neck (e.g., nasopharyngeal carcinoma,        Endolymphatic Sac Tumor (ELST), epidermoid carcinoma, laryngeal        cancers including squamous cell carcinoma (SCC) (e.g., glottic        carcinoma, supraglottic carcinoma, subglottic carcinoma,        transglottic carcinoma), carcinoma in situ, verrucous, spindle        cell and basaloid SCC, undifferentiated carcinoma, laryngeal        adenocarcinoma, adenoid cystic carcinoma, neuroendocrine        carcinomas, laryngeal sarcoma), head and neck paragangliomas        (e.g., carotid body, jugulotympanic, vagal); thymus (e.g.,        thymoma); heart (e.g., cardiac myxoma); lung (e.g., small cell        carcinoma (SCLC), non-small cell lung carcinoma (NSCLC),        including squamous cell carcinoma (SCC), adenocarcinoma and        large cell carcinoma, carcinoids (typical or atypical),        carcinosarcomas, pulmonary blastomas, giant cell carcinomas,        spindle cell carcinomas, pleuropulmonary blastoma); lymph (e.g.,        lymphomas, including Hodgkin's lymphoma, non-Hodgkin's lymphoma,        Epstein-Barr virus (EBV)-associated lymphoproliferative        diseases, including B cell lymphomas and T cell lymphomas (e.g.,        Burkitt lymphoma, large B cell lymphoma, diffuse large B-cell        lymphoma (DLBCL), mantle cell lymphoma, indolent B-cell        lymphoma, low grade B cell lymphoma, fibrin-associated diffuse        large cell lymphoma; primary effusion lymphoma; plasmablastic        lymphoma; extranodal NK/T cell lymphoma, nasal type; peripheral        T cell lymphoma, cutaneous T cell lymphoma, angioimmunoblastic T        cell lymphoma; follicular T cell lymphoma; systemic T cell        lymphoma), lymphangioleiomyomatosis); central nervous system        (CNS) (e.g., gliomas including astrocytic tumors (e.g.,        pilocytic astrocytoma, pilomyxoid astrocytoma, subependymal        giant cell astrocytoma, pleomorphic xanthoastrocytoma, diffuse        astrocytoma, fibrillary astrocytoma, gemistocytic astrocytoma,        protoplasmic astrocytoma, anaplastic astrocytoma, glioblastoma        (e.g., giant cell glioblastoma, gliosarcoma, glioblastoma        multiforme) and gliomatosis cerebri), oligodendroglial tumors        (e.g., oligodendroglioma, anaplastic oligodendroglioma),        oligoastrocytic tumors (e.g., oligoastrocytoma, anaplastic        oligoastrocytoma), ependymal tumors (e.g., subependymom,        myxopapillary ependymoma, ependymomas (e.g., cellular,        papillary, clear cell, tanycytic), anaplastic ependymoma), optic        nerve glioma, and non-gliomas (e.g., choroid plexus tumors,        neuronal and mixed neuronal-glial tumors, pineal region tumors,        embryonal tumors, medulloblastoma, meningeal tumors, primary CNS        lymphomas, germ cell tumors, pituitary adenomas, cranial and        paraspinal nerve tumors, stellar region tumors), neurofibroma,        meningioma, peripheral nerve sheath tumors, peripheral        neuroblastic tumours (including without limitation        neuroblastoma, ganglioneuroblastoma, ganglioneuroma), trisomy 19        ependymoma); neuroendocrine tissues (e.g., paraganglionic system        including adrenal medulla (pheochromocytomas) and extra-adrenal        paraganglia ((extra-adrenal) paragangliomas); skin (e.g., clear        cell hidradenoma, cutaneous benign fibrous histiocytomas,        cylindroma, hidradenoma, melanoma (including cutaneous melanoma,        mucosal melanoma), pilomatricoma, Spitz tumors); and soft        tissues (e.g., aggressive angiomyxoma, alveolar        rhabdomyosarcoma, alveolar soft part sarcoma, angiofibroma,        angiomatoid fibrous histiocytoma, synovial sarcoma, biphasic        synovial sarcoma, clear cell sarcoma, dermatofibrosarcoma        protuberans, desmoid-type fibromatosis, small round cell tumor,        desmoplastic small round cell tumor, elastofibroma, embryonal        rhabdomyosarcoma, Ewing's tumors/primitive neurectodermal tumors        (PNET), extraskeletal myxoid chondrosarcoma, extraskeletal        osteosarcoma, paraspinal sarcoma, inflammatory myofibroblastic        tumor, lipoblastoma, lipoma, chondroid lipoma,        liposarcoma/malignant lipomatous tumors, liposarcoma, myxoid        liposarcoma, fibromyxoid sarcoma, lymphangioleiomyoma, malignant        myoepithelioma, malignant melanoma of soft parts, myoepithelial        carcinoma, myoepithelioma, myxoinflammatory fibroblastic        sarcoma, undifferentiated sarcoma, pericytoma, rhabdomyosarcoma,        non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), soft tissue        leiomyosarcoma, undifferentiated sarcoma, well-differentiated        liposarcoma.    -   Embodiment 191. The method of any one of embodiments 156-184,        wherein the cancer is selected from the group consisting of a        melanoma, a gastric cancer, a triple-negative breast cancer        (TNBC), a non-small cell lung cancer (NSCLC), a rectal        adenocarcinoma, a colorectal cancer, a renal cell carcinoma, an        ovarian cancer, a prostate cancer, an oral squamous cell        carcinoma (SCC), a head and neck squamous cell carcinoma        (HNSCC), a urothelial bladder cancer, a glioblastoma (GBM), a        meningioma, adrenal cancer, and an endometrial cancer.    -   Embodiment 192. The antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition of any one of        embodiments 1-146, 148-151, 153, or 154 for use in the treatment        of cancer.    -   Embodiment 193. The antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition for use of        embodiment 192, wherein the cancer is selected from the group        consisting of a solid tumor, a hematological cancer, and a        metastatic lesion.    -   Embodiment 194. The antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition for use of        embodiment 192, wherein the solid tumor is selected from the        group consisting of a sarcoma, a fibroblastic sarcoma, a        carcinoma, and an adenocarcinoma.    -   Embodiment 195. The antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition for use of        embodiment 192, wherein the hematological cancer is selected        from the group consisting of a leukemia, a lymphoma, and a        myeloma.    -   Embodiment 196. The antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition for use of any one        of embodiments 192-195, wherein the cancer is selected from the        group consisting of a lung cancer, a melanoma, a renal cancer, a        liver cancer, a myeloma, a prostate cancer, a breast cancer, an        ovarian cancer, a colorectal cancer, a pancreatic cancer, a head        and neck cancer, an anal cancer, a gastro-esophageal cancer, a        mesothelioma, a nasopharyngeal cancer, a thyroid cancer, a        cervical cancer, an epithelial cancer, a peritoneal cancer, a        lymphoproliferative disease, an acute lymphoblastic leukemia        (ALL), an acute myelogenous leukemia (AML), a chronic        lymphocytic leukemia (CLL), a chronic myelogenous leukemia        (CML), a chronic myelomonocytic leukemia (CMML), a hairy cell        leukemia, a B cell lymphoma, a diffuse large B-cell lymphoma        (DLBCL), an activated B-cell like (ABC) diffuse large B cell        lymphoma, a germinal center B cell (GCB) diffuse large B cell        lymphoma, a mantle cell lymphoma, a Hodgkin lymphoma, a        non-Hodgkin lymphoma, a relapsed non-Hodgkin lymphoma, a        refractory non-Hodgkin lymphoma, a recurrent follicular        non-Hodgkin lymphoma, a Burkitt lymphoma, a small lymphocytic        lymphoma, a follicular lymphoma, a lymphoplasmacytic lymphoma,        and an extranodal marginal zone lymphoma.    -   Embodiment 197. The antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition of any one of        embodiments 192-195, wherein the cancer is selected from the        group consisting of a melanoma, a gastric cancer, a        triple-negative breast cancer (TNBC), a non-small cell lung        cancer (NSCLC), a rectal adenocarcinoma, a colorectal cancer, a        renal cell carcinoma, an ovarian cancer, a prostate cancer, an        oral squamous cell carcinoma (SCC), a head and neck squamous        cell carcinoma (HNSCC), a urothelial bladder cancer, a        glioblastoma (GBM), a meningioma, adrenal cancer, and an        endometrial cancer.    -   Embodiment 198. Use of the antibody, polynucleotide, vector,        lipid nanoparticle, or pharmaceutical composition of any one of        embodiments 1-146, 148-151, 153, or 154 for the preparation of a        medicament for treating cancer.    -   Embodiment 199. The use of embodiment 198, wherein the cancer is        selected from the group consisting of a solid tumor, a        hematological cancer, and a metastatic lesion.    -   Embodiment 200. The use of embodiment 199, wherein the solid        tumor is selected from the group consisting of a sarcoma, a        fibroblastic sarcoma, a carcinoma, and an adenocarcinoma.    -   Embodiment 201. The use of embodiment 199, wherein the        hematological cancer is selected from the group consisting of a        leukemia, a lymphoma, and a myeloma.    -   Embodiment 202. The use of any one of embodiments 198-201,        wherein the cancer is selected from the group consisting of a        lung cancer, a melanoma, a renal cancer, a liver cancer, a        myeloma, a prostate cancer, a breast cancer, an ovarian cancer,        a colorectal cancer, a pancreatic cancer, a head and neck        cancer, an anal cancer, a gastro-esophageal cancer, a        mesothelioma, a nasopharyngeal cancer, a thyroid cancer, a        cervical cancer, an epithelial cancer, a peritoneal cancer, a        lymphoproliferative disease, an acute lymphoblastic leukemia        (ALL), an acute myelogenous leukemia (AML), a chronic        lymphocytic leukemia (CLL), a chronic myelogenous leukemia        (CML), a chronic myelomonocytic leukemia (CMML), a hairy cell        leukemia, a B cell lymphoma, a diffuse large B-cell lymphoma        (DLBCL), an activated B-cell like (ABC) diffuse large B cell        lymphoma, a germinal center B cell (GCB) diffuse large B cell        lymphoma, a mantle cell lymphoma, a Hodgkin lymphoma, a        non-Hodgkin lymphoma, a relapsed non-Hodgkin lymphoma, a        refractory non-Hodgkin lymphoma, a recurrent follicular        non-Hodgkin lymphoma, a Burkitt lymphoma, a small lymphocytic        lymphoma, a follicular lymphoma, a lymphoplasmacytic lymphoma,        and an extranodal marginal zone lymphoma.    -   Embodiment 203. The use of any one of embodiments 198-201,        wherein the cancer is selected from the group consisting of a        melanoma, a gastric cancer, a triple-negative breast cancer        (TNBC), a non-small cell lung cancer (NSCLC), a rectal        adenocarcinoma, a colorectal cancer, a renal cell carcinoma, an        ovarian cancer, a prostate cancer, an oral squamous cell        carcinoma (SCC), a head and neck squamous cell carcinoma        (HNSCC), a urothelial bladder cancer, a glioblastoma (GBM), a        meningioma, adrenal cancer, and an endometrial cancer.    -   Embodiment 204. The antibody, polynucleotide, vector, lipid        nanoparticle, or pharmaceutical composition of any one of        embodiments 1-146, 148-151, 153, or 154, for use in the method        of any one of embodiments 156-191.    -   Embodiment 205. Use of the antibody, polynucleotide, vector,        lipid nanoparticle, or pharmaceutical composition of any one of        embodiments 1-146, 148-151, 153, or 154 in the method of any one        of embodiments 156-191.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of graphs showing the binding of anti-CD73 antibodies(BA013, BA014, BA015, or a reference antibody (“RA001”)), or an isotypecontrol antibody, to Chinese hamster ovarian (CHO) cells ectopicallyexpressing mouse, cynomolgus, or human CD73 (left to right in thefigure), as measured by flow cytometry. The median fluorescenceintensity (MFI) values are plotted against antibody concentrations.

FIGS. 2A-2B are a set of graphs showing the extent of inhibition of CD73enzymatic activity by anti-CD73 antibodies (BA013, BA014, BA015, orRA001) or an isotype control (FIG. 2A), or by anti-CD73 antibody BA015or the isotype control (FIG. 2B). The percent activity of CD73ectopically expressed on the surface of Jurkat cells (FIG. 2A) or CHOcells (FIG. 2B) is plotted as luminescence levels (measured in RelativeLight Unit (RLU)) relative to the levels observed in the absence ofantibody.

FIGS. 3A-3C are a set of graphs and images showing internalization ofanti-CD73 antibodies and antibody-mediated internalization of CD73. FIG.3A shows results of a cytotoxicity assay measuring antibodyinternalization by incubating CD73-expressing cells with test antibodies(BA015, RA001, or a second reference antibody (“RA002”)), isotypecontrol, or no antibody, and a secondary antibody conjugated withcytotoxic monomethyl auristatin E (MMAE). The levels of antibodyinternalization are plotted as the viability of CD73-expressing cells(normalized to the viability of control cells that were not treated withany primary or secondary antibodies (“NT”)) against antibodyconcentration. FIG. 3B shows a series of representative images from aCD73 internalization assay, wherein cells expressing a HaloTag®-CD73fusion protein are detected by confocal fluorescence microscopy afterincubation of the cells with a HaloTag® Alexa Fluor™ 488 ligand thatstains the fusion protein and treatment with anti-CD73 or controlantibodies. FIG. 3C is a graph showing quantification of intracellularHaloTag®-CD73 from four images acquired in the FITC channel (to detectHaloTag® Alexa Fluor™ 488) every hour for each condition over a courseof 6 hours.

FIGS. 4A-4P are a set of graphs showing binding of an isotope control,RA001, RA002, BA015, and humanized anti-CD73 antibodies BA019-BA030 toCHO cells ectopically expressing cynomolgus CD73, as measured by flowcytometry. The MFI values are plotted against antibody concentrations toshow binding curves.

FIGS. 5A-5P are a set of graphs showing binding of an isotope control,RA001, RA002, BA015, and humanized anti-CD73 antibodies BA019-BA030 toCHO cells ectopically expressing human CD73, as measured by flowcytometry. A negative control without any antibody, designated “noantibody,” was used for comparison in FIG. 5A. The MFI values areplotted against antibody concentrations to show binding curves.

FIGS. 6A-6P are a set of graphs showing the extent of inhibition of CD73enzymatic activity by an isotope control, RA001, RA002, BA015, andhumanized anti-CD73 antibodies BA019-BA030. The percent activity of CD73ectopically expressed on the surface of Jurkat cells is plotted asluminescence levels (measured in RLU) relative to the levels in theabsence of antibody.

FIGS. 7A-7P are a set of graphs showing the extent of inhibition of CD73enzymatic activity by an isotope control, RA001, RA002, BA015, andhumanized anti-CD73 antibodies BA019-BA030. The percent activity ofimmobilized, recombinant CD73 is plotted as luminescence levels(measured in RLU) relative to the levels in the absence of antibody.

FIGS. 8A-8M are a set of graphs showing the results of a cytotoxicityassay that measures antibody internalization by incubatingCD73-expressing cells with no antibody, isotope control, BA015, or ahumanized anti-CD73 antibody, and a secondary antibody conjugated withMMAE. The levels of antibody internalization are plotted as theviability of the CD73-expressing cells (normalized to the viability ofcontrol cells that were not treated with any primary or secondaryantibodies (“NT”)) against antibody concentrations.

FIGS. 9A-9E are a set of graphs showing the abilities of an anti-CD73antibody (BA012) or an anti-CD73-TGFβ trap fusion protein (BA035), butnot isotype controls (an isotype antibody control and a fusion proteincontrol comprising such isotype antibody and the TGFβ trap), to bindCD73 and inhibit CD73 enzymatic activity. FIGS. 9A and 9B show bindingof BA012 or BA035, but not isotype controls, to CHO cells ectopicallyexpressing mouse CD73 in the absence (FIG. 9A) or presence (FIG. 9B) ofTGFβ1. The levels of binding, as measured by flow cytometry, are plottedas absolute MFI values (FIG. 9A) or as percentage MFI values relative tothe MFI value of each test condition in the absence of TGFβ1 (FIG. 9B).FIGS. 9C-9E show inhibition of CD73 enzymatic activity by BA012 orBA035, but not the isotype control, in the absence (FIGS. 9C and 9D) orpresence (FIG. 9E) of TGFβ1. The percent activity of CD73 ectopicallyexpressed on the surface of CHO cells is plotted as luminescence levels(measured in RLU) relative to the levels in the absence of antibody.

FIGS. 10A and 10B are a set of graphs showing antagonism of TGFβ-inducedsignaling by BA012 or BA035, but not isotype controls. In FIG. 10A,relative luminescence levels, which correlate with TGFβ1-inducedexpression of luciferase in HEK293-SMAD reporter cells, are plotted aspercentage values relative to the luminescence levels in the absence ofantibody. In FIG. 10B, the expression levels of red fluorescent protein(RFP), which correlate with TGFβ1-induced expression of Vimentin inA549-Vimentin-RFP cells, are plotted as absolute fluorescence valuesminus the background fluorescence levels in the absence of antibody orTGFβ1.

FIGS. 11A and 11B are a set of graphs showing the inhibition of tumorgrowth and metastasis by anti-CD73 antibodies (an isotope control,BA012, and a reference antibody comprising the variable domain of RA001and a murine IgG2a-N297A constant domain, designated RA001-IgG2a-N297A).FIG. 11A shows mean calculated tumor volumes of 8-10 mice/groupimplanted subcutaneously with CT26 tumor cells and treated with theindicated antibodies. FIG. 11B shows the numbers of tumor nodules in thelungs of each mouse implanted intravenously with EMT6 tumor cells andtreated with BA012 or isotype control antibody.

FIGS. 12A and 12B are a set of graphs showing inhibition of tumor growthby BA012, BA035, or isotype controls. FIG. 12A shows mean calculatedtumor volumes of 5-7 mice/group implanted with TC1 tumor cellssubcutaneously and treated with the indicated antibodies. FIG. 12B showscalculated tumor volumes of 8-10 mice/group implanted with EMT6 tumorcells subcutaneously and treated with the indicated antibodies.

FIGS. 13A and 13B are a set of graphs showing the binding of ananti-CD73 antibody (BA025), an anti-CD73-TGFβ trap fusion protein(BA037), an isotype control antibody, or an isotype-TGFβ trap to CD8⁺ Tcells isolated from PBMCs of either human (FIG. 13A) or cynomolgusmonkey (FIG. 13B), as measured by flow cytometry. The median MFI valuesare plotted against antibody concentrations.

FIGS. 14A-14D are a set of graphs showing the extent of inhibition ofrecombinant human CD73 enzymatic activity in a biochemical assay by ananti-CD73 antibody (BA025), an anti-CD73-TGFβ trap fusion protein(BA037), an isotype control antibody, or an isotype-TGFβ trap. Theresults of two experiments are shown. The activity of immobilizedplate-bound CD73 (FIGS. 14A and 14B) or soluble CD73 (FIGS. 14C and 14D)is plotted as luminescence levels (measured in RLU) relative to thelevels in the absence of antibody.

FIGS. 15A-15D are a set of graphs showing the extent of inhibition ofrecombinant cynomolgus monkey CD73 enzymatic activity in a biochemicalassay by an anti-CD73 antibody (BA025), an anti-CD73-TGFβ trap fusionprotein (BA037), an isotype control antibody, or an isotype-TGFβ trap.The results of two experiments are shown. The activity of immobilizedplate-bound CD73 (FIGS. 15A and 15B) or soluble CD73 (FIGS. 15C and 15D)is plotted as luminescence levels (measured in RLU) relative to thelevels in the absence of antibody.

FIGS. 16A and 16B are a set of graphs showing the extent of inhibitionof CD73 enzymatic activity by an anti-CD73 antibody (BA025), ananti-CD73-TGFβ trap fusion protein (BA037), an isotype control antibody,or an isotype-TGFβ trap. The percent activity of CD73 ectopicallyexpressed on the surface of Jurkat cells in two experiments (FIG. 16Aand FIG. 16B) is plotted as luminescence levels (measured in RLU)relative to the levels in the absence of antibody.

FIGS. 17A-17C are a set of graphs showing antagonism of TGFβ-inducedsignaling in an HEK293 SMAD-luciferase reporter assay by an anti-CD73antibody (BA025), an anti-CD73-TGFβ trap fusion protein (BA037), anisotype control antibody, or an isotype-TGFβ trap. Each TGFβ isoform wastested separately. The TGFβ-induced SMAD signaling activity (whichcorrelates with luciferase activity in the HEK293-SMAD-luciferase cells)is plotted as luminescence levels (measured in RLU) relative to thelevels in the absence of antibody.

FIGS. 18A-18I are a set of graphs showing antagonism of TGFβ-inducedsignaling in an A549-Vimentin-RFP reporter cell assay by an anti-CD73antibody (BA025), an anti-CD73-TGFβ trap fusion protein (BA037), anisotype control antibody, or an isotype-TGFβ trap. Each TGFβ isoform wastested separately. The results of three experiments are shown. Twoexperiments (FIGS. 18A-18C and 18D-18F) were conducted at roomtemperature. One experiment (FIGS. 18G-18I) was conducted at 37° C. TheTGFβ-induced signaling activity (which correlate with RFP expression inthe A549-Vimentin-RFP cells) is plotted as MFI of RFP relative to thelevels in the absence of antibody.

FIGS. 19A-19D are a set of graphs showing internalization of anti-CD73antibodies and antibody-mediated internalization of CD73. The graphsshow results of a cytotoxicity assay measuring antibody internalizationby incubating CD73-expressing cells with an anti-CD73 antibody (BA025),an anti-CD73-TGFβ trap fusion protein (BA037), an isotype controlantibody, or no antibody, in each case together with a secondaryantibody conjugated with the cytotoxic agent DM1. The levels of antibodyinternalization are measured by cell death which is indicated by a lossof fluorescence intensity (measured in RLU). The data are plotted as theRLU of CD73-expressing cells against antibody concentration. Data fromtwo experiments are shown.

FIGS. 20A and 20B are a set of graphs showing ability of an anti-CD73antibody (BA025), an anti-CD73-TGFβ trap fusion protein (BA037), anisotype control antibody, an isotype-TGFβ trap, and an Fc-competentanti-CD73 to activate FcγR receptors on Jurkat cells containing anNFAT-luciferase reporter construct and expressing either FcγRIIA (H131)(FIG. 20A) or Jurkat FcγRIIIA (V158) (FIG. 20B), in each case in thepresence of CHO cells expressing CD73. FcγR receptor activation isplotted as luminescence levels (measured in RLU) relative to the levelsin the absence of antibody.

FIG. 21 is a graph showing the inhibition of metastasis by BA035. Thegraph shows the numbers of tumor nodules in the lungs of each mouseimplanted intravenously with EMT6 tumor cells and treated with BA012,BA035 or isotype control antibody.

FIG. 22 is a graph showing inhibition of tumor growth by BA012, BA035,or isotype controls. The graph shows mean calculated tumor volumes from5-7 mice/group implanted with LLC tumor cells and treated with theindicated antibodies.

FIG. 23 is a graph showing inhibition of tumor growth by BA012, BA035,or isotype controls. The graph shows mean calculated tumor volumes of8-10 mice/group implanted with Detroit562 tumor cells subcutaneously andtreated with the indicated antibodies.

FIGS. 24A-24D are a set of graphs showing inhibition of tumor growth byBA012, BA035, or isotype controls, administered alone or in combinationwith either an anti-PD-1 antibody or doxorubicin. The graphs showcalculated tumor volumes of 6-9 mice/group implanted subcutaneously withEMT6 tumor cells and treated with the indicated antibodies. FIG. 24Ashows data from mice that did not receive a combination treatment. FIG.24B shows data from mice that received anti-PD-1 in addition to theindicated antibodies. FIGS. 24C and 24D show data from mice thatreceived doxorubicin in addition to the indicated antibodies. The graphin FIG. 24D shows the same data as the graph in FIG. 24C, except thatthe isotype control data has been removed and the x-axis has beenreplotted to better separate data from the different treatment groups.

FIG. 25 is a graph showing the inhibition of tumor growth by BA012,BA035, or isotype controls in combination with etoposide, or byetoposide alone. The graph shows mean calculated tumor volumes of 5-8mice/group implanted subcutaneously with CT26 tumor cells and treatedwith the indicated antibodies.

5. DETAILED DESCRIPTION

The instant disclosure provides antibodies that specifically bind toCD73 (e.g., human CD73, cynomolgus CD73, or mouse CD73) and antagonizeCD73 function, e.g., the enzymatic activity to promote AMP conversion toadenosine. Also provided are pharmaceutical compositions comprisingthese antibodies, nucleic acids encoding these antibodies, expressionvectors and host cells for making these antibodies, and methods oftreating a subject using these antibodies. The antibodies disclosedherein are particularly useful for boosting immune responses to a tumorantigen, and hence, are useful for treating cancer in a subject. Allinstances of “isolated antibodies” described herein are additionallycontemplated as antibodies that may be, but need not be, isolated. Allinstances of “isolated polynucleotides” described herein areadditionally contemplated as polynucleotides that may be, but need notbe, isolated. All instances of “antibodies” described herein areadditionally contemplated as antibodies that may be, but need not be,isolated. All instances of “polynucleotides” described herein areadditionally contemplated as polynucleotides that may be, but need notbe, isolated.

5.1 Definitions

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., upto 5% to 10% below) the value or range remain within the intendedmeaning of the recited value or range.

As used herein, the term “CD73” (also known as ecto-5′-nucleotidase,ecto-5′-NT, 5′-NT, and NT5E) refers to a protein that in humans isencoded by the NT5E gene. As used herein, the term “human CD73” refersto a CD73 protein encoded by a human NT5E gene (e.g., a wild-type humanNT5E gene) or an extracellular domain of such a protein. Exemplarywild-type human CD73 proteins are provided by GenBank™ accession numbersNP_002517.1, NP_001191742.1, AAH65937, and EAW48635. For this definitionand the definitions of other proteins herein, the skilled worker willappreciate that a protein encoded by a gene can be in its mature form,e.g., with its signal peptide removed. Exemplary amino acid sequences ofan immature human CD73 protein are provided as SEQ ID NOs: 99, 100, 129,and 130. Exemplary amino acid sequence of a mature human CD73 proteinare provided as amino acid residues Trp27-Gln574 of SEQ ID NO: 99 andamino acid residues Trp27-Gln524 of SEQ ID NO: 100. Exemplary amino acidsequences of an extracellular domain of a mature human CD73 protein areprovided as amino acid residues Trp27-Lys547 of SEQ ID NO: 99 andTrp27-Lys497 of SEQ ID NO: 100.

As used herein, the terms “transforming growth factor beta” and “TGFβ”refer to any of the TGFβ family proteins that in human are encoded bythe TGFB1, TGFB2, and TGFB3 genes. As used herein, the term “humanTGFβ1” refers to a TGFβ1 protein encoded by a human TGFB1 gene (e.g., awild-type human TGFB1 gene). An exemplary wild-type human TGFβ1 proteinis provided by GenBank™ accession number NP_000651.3. As used herein,the term “human TGFβ2” refers to a TGFβ2 protein encoded by a humanTGFB2 gene (e.g., a wild-type human TGFB2 gene). Exemplary wild-typehuman TGFβ2 proteins are provided by GenBank™ accession numbersNP_001129071.1 and NP_003229.1. As used herein, the term “human TGFβ3”refers to a TGFβ3 protein encoded by a human TGFB3 gene (e.g., awild-type human TGFB3 gene). Exemplary wild-type human TGFβ3 proteinsare provided by GenBank™ accession numbers NP_003230.1, NP_001316868.1,and NP_001316867.1.

As used herein, the terms “transforming growth factor beta receptor” and“TGFβ receptor” refer to any of the TGFβ receptor family proteins thatin human are encoded by the TGFBR1, TGFBR2, and TGFBR3 genes. As usedherein, the term “human TGFβR1” refers to a TGFβR1 protein encoded by ahuman TGFBR1 gene (e.g., a wild-type human TGFBR1 gene). Exemplarywild-type human TGFβR1 proteins are provided by GenBank™ accessionnumbers NP_004603.1, NP_001124388.1, and NP_001293139.1. As used herein,the term “human TGFβ2” refers to a TGFβ2 protein encoded by a humanTGFB2 gene (e.g., a wild-type human TGFB2 gene). Exemplary wild-typehuman TGFβ2 proteins are provided by GenBank™ accession numbersNP_001129071.1 and NP_003229.1. As used herein, the term “human TGFβ3”refers to a TGFβ3 protein encoded by a human TGFB3 gene (e.g., awild-type human TGFB3 gene). Exemplary wild-type human TGFβ3 proteinsare provided by GenBank™ accession numbers NP_003230.1, NP_001316868.1,and NP_001316867.1.

As used herein, the terms “vascular endothelial growth factor” and“VEGF” refer to any of the VEGF family proteins that in human areencoded by the VEGFA, VEGFB, VEGFC, and VEGFD genes. As used herein, theterm “human VEGF-A” refers to a VEGF-A protein encoded by a human VEGFAgene (e.g., a wild-type human VEGFA gene). Exemplary wild-type humanVEGF-A proteins are provided by GenBank™ accession numbersNP_001020537.2, NP_001020538.2, NP 001020539.2, NP_001020540.2, NP001020541.2, NP_001028928.1, NP_001165093.1, NP 001165094.1, NP001165095.1, NP 001165096.1, NP_001165097.1, NP 001165098.1, NP001165099.1, NP 001165100.1, NP_001165101.1, NP_001191313.1,NP_001191314.1, NP_001273973.1, NP_001303939.1, and NP_003367.4. As usedherein, the term “human VEGF-B” refers to a VEGF-B protein encoded by ahuman VEGFB gene (e.g., a wild-type human VEGFB gene). Exemplarywild-type human VEGF-B proteins are provided by GenBank™ accessionnumbers NP 001230662.1 and NP 003368.1. As used herein, the term “humanVEGF-C” refers to a VEGF-C protein encoded by a human VEGFC gene (e.g.,a wild-type human VEGFC gene). Exemplary wild-type human VEGF-C proteinsare provided by GenBank™ accession number NP_005420.1. As used herein,the term “human VEGF-D” refers to a VEGF-D protein encoded by a humanVEGFD gene (e.g., a wild-type human VEGFD gene). Exemplary wild-typehuman VEGF-D proteins are provided by GenBank™ accession numberNP_004460.1.

As used herein, the terms “vascular endothelial growth factor receptor”and “VEGFR” refer to any of the VEGF receptor family proteins that inhuman are encoded by the FLT1 (also known as VEGFR1, KDR (also known asVEGFR2), and FLT4 (also known as VEGFR3) genes. As used herein, the term“human VEGFR1” refers to a VEGFR1 protein encoded by a human FLT1 gene(e.g., a wild-type FLT1 gene). Exemplary wild-type human VEGFR1 proteinsare provided by GenBank™ accession numbers NP_002010.2, NP_001153392.1,NP_001153502.1, and NP_001153503.1. As used herein, the term “humanVEGFR2” refers to a VEGFR2 protein encoded by a human KDR gene (e.g., awild-type human KDR gene). An exemplary wild-type human VEGFR2 proteinis provided by GenBank™ accession number NP_002244.1. As used herein,the term “human VEGFR3” refers to a VEGFR3 protein encoded by a humanFLT4 gene (e.g., a wild-type human FLT4 gene). Exemplary wild-type humanVEGFR3 proteins are provided by GenBank™ accession numbers NP_891555.2,NP_002011.2, and NP_001341918.1.

As used herein, the terms “antibody” and “antibodies” includefull-length antibodies, antigen-binding fragments of full-lengthantibodies, and molecules comprising antibody CDRs, VH regions, and/orVL regions. Examples of antibodies include, without limitation,monoclonal antibodies, recombinantly produced antibodies, monospecificantibodies, multispecific antibodies (including bispecific antibodies),human antibodies, humanized antibodies, chimeric antibodies,immunoglobulins, synthetic antibodies, tetrameric antibodies comprisingtwo heavy chain and two light chain molecules, an antibody light chainmonomer, an antibody heavy chain monomer, an antibody light chain dimer,an antibody heavy chain dimer, an antibody light chain- antibody heavychain pair, intrabodies, heteroconjugate antibodies, antibody-drugconjugates, single domain antibodies, monovalent antibodies, singlechain antibodies or single-chain Fvs (scFv), camelized antibodies,affibodies, Fab fragments, F(ab′)₂ fragments, disulfide-linked Fvs(sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-anti-Id antibodies), and antigen-binding fragments of any of theabove, and conjugates or fusion proteins comprising any of the above. Incertain embodiments, antibodies described herein refer to polyclonalantibody populations. Antibodies can be of any type (e.g., IgG, IgE,IgM, IgD, IgA or IgY), any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ orIgA₂), or any subclass (e.g., IgG_(2a) or IgG_(2b)) of immunoglobulinmolecule. In certain embodiments, antibodies described herein are IgGantibodies, or a class (e.g., human IgG₁ or IgG₄) or subclass thereof.In a specific embodiment, the antibody is a humanized monoclonalantibody. In another specific embodiment, the antibody is a humanmonoclonal antibody. In certain embodiments, an antibody, as describedherein, comprises a full-length antibody (e.g., a full-length antibodythat specifically binds to CD73), or an antigen-binding fragmentthereof, linked to a ligand binding moiety that specifically binds to amolecule other than CD73 (e.g., a ligand binding moiety thatspecifically binds to TGFβ or VEGF, e.g., a ligand binding moietycomprising one or more ectodomains of a TGFβ receptor or VEGF receptor).

As used herein, the terms “VH region” and “VL region” refer,respectively, to single antibody heavy and light chain variable regions,comprising FR (Framework Regions) 1, 2, 3 and 4 and CDR (ComplementarityDetermining Regions) 1, 2 and 3 (see Kabat et al., (1991) Sequences ofProteins of Immunological Interest (NIH Publication No. 91-3242,Bethesda), which is herein incorporated by reference in its entirety).

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), by Chothia et al., J. Mol. Biol.196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745(1996), all of which are herein incorporated by reference in theirentireties, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. In certainembodiments, the term “CDR” is a CDR as defined by MacCallum et al., J.Mol. Biol. 262:732-745 (1996) and Martin A. “Protein Sequence andStructure Analysis of Antibody Variable Domains,” in AntibodyEngineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439,Springer-Verlag, Berlin (2001). In certain embodiments, the term “CDR”is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616(1977) and Kabat et al., Sequences of protein of immunological interest.(1991). In certain embodiments, heavy chain CDRs and light chain CDRs ofan antibody are defined using different conventions. In certainembodiments, heavy chain CDRs and/or light chain CDRs are defined byperforming structural analysis of an antibody and identifying residuesin the variable region(s) predicted to make contact with an epitoperegion of a target molecule (e.g., human and/or cynomolgus CD73). CDRH1,CDRH2 and CDRH3 denote the heavy chain CDRs, and CDRL1, CDRL2 and CDRL3denote the light chain CDRs.

As used herein, the term “framework (FR) amino acid residues” refers tothose amino acids in the framework region of an antibody variableregion. The term “framework region” or “FR region” as used herein,includes the amino acid residues that are part of the variable region,but are not part of the CDRs (e.g., using the Kabat or MacCallumdefinition of CDRs).

As used herein, the terms “variable region” and “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 120amino acids or 110 to 125 amino acids in the mature heavy chain andabout 90 to 115 amino acids in the mature light chain, which differextensively in sequence among antibodies and are used in the binding andspecificity of a particular antibody for its particular antigen. Thevariability in sequence is concentrated in those regions calledcomplementarity determining regions (CDRs) while the more highlyconserved regions in the variable domain are called framework regions(FR). Without wishing to be bound by any particular mechanism or theory,it is believed that the CDRs of the light and heavy chains are primarilyresponsible for the interaction and specificity of the antibody withantigen. In certain embodiments, the variable region is a human variableregion. In certain embodiments, the variable region comprises rodent ormurine CDRs and human framework regions (FRs). In particularembodiments, the variable region is a primate (e.g., non-human primate)variable region. In certain embodiments, the variable region comprisesrodent or murine CDRs and primate (e.g., non-human primate) frameworkregions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

As used herein, the terms “constant region” and “constant domain” areinterchangeable and are common in the art. The constant region is anantibody portion, e.g., a carboxyl terminal portion of a light and/orheavy chain which is not directly involved in binding of an antibody toantigen but which can exhibit various effector functions, such asinteraction with an Fc receptor (e.g., Fc gamma receptor). The constantregion of an immunoglobulin molecule generally has a more conservedamino acid sequence relative to an immunoglobulin variable domain.

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG, and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃, and IgG₄.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art. In specific embodiments,the light chain is a human light chain.

As used herein, the term “EU numbering system” refers to the EUnumbering convention for the constant regions of an antibody, asdescribed in Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969) and Kabat et al, Sequences of Proteins of Immunological Interest,U.S. Dept. Health and Human Services, 5th edition, 1991, each of whichis herein incorporated by reference in its entirety.

As used herein, the term “binding moiety” refers to a moiety that hasthe ability to specifically bind to a target molecule or complex. Thebinding moiety can comprise a small molecule, peptide, modified peptide(e.g., peptides having non-natural amino acid residues, and/or stapledpeptides), polypeptide, protein, antibody, antibody fragment, scFv,Fc-containing polypeptide, fusion antibody, ligand, aptamer, nucleicacid, variants thereof, or any combination thereof. In certainembodiments, the binding moiety comprises a domain of a natural proteinor a variant thereof, wherein the natural protein binds to the targetmolecule or complex via the domain. For example, the term “TGFβ-bindingmoiety” refers to a moiety that specifically binds to one or moreproteins of the TGFβ family (e.g., TGFβ 1, TGFβ2, or TGFβ3), and incertain embodiments can comprise an extracellular domain of a TGFβreceptor (e.g., TGFβR1, TGFβR2, or TGFβR3). The term “VEGF-bindingmoiety” refers to a moiety that specifically binds to one or moreproteins of the VEGF family (e.g., VEGF-A, VEGF-B, VEGF-C, or VEGF-D),and in certain embodiments can comprise an extracellular domain of aVEGF receptor (e.g., VEGFR1, VEGFR2, or VEGFR3).

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured and/or expressedin a number of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D)), and equilibrium associationconstant (K_(A)). The K_(D) is calculated from the quotient ofk_(off)/k_(on), whereas K_(A) is calculated from the quotient ofk_(on)/k_(off). k_(on) refers to the association rate constant of, e.g.,an antibody to an antigen, and k_(off) refers to the dissociation rateconstant of, e.g., an antibody to an antigen. The k_(on) and k_(off) canbe determined by techniques known to one of ordinary skill in the art,such as BIAcore® or KinExA. As used herein, a “lower affinity” refers toa larger K_(D).

As used herein, the term “maximal binding” refers to the maximal signal(expressed in resonance units (RU)) observed for the binding of anantigen (e.g., a CD73 protein) at a given concentration to an antibody(e.g., an anti-CD73 antibody) in a surface plasmon resonance bindingassay (e.g., a surface plasmon resonance binding assay described insection 6.5.2 herein).

As used herein in the context of binding affinity, the binding affinityof a test antibody for a first antigen is “substantially reduced”relative to the binding affinity of the test antibody for a secondantigen if the binding affinity of the test antibody for the firstantigen is reduced by at least 30%, 40%, 50%, 60%, 70%, 80% or 90%relative to the binding affinity of the test antibody for the secondantigen, e.g., in a given experiment, or using mean values from multipleexperiments, as assessed by, e.g., a binding affinity assay disclosedherein. As used herein in the context of maximal binding, the bindingbetween a test antibody and a first antigen is “substantially reduced”relative to the maximal binding between the test antibody and a secondantigen if the maximal binding between the test antibody and the firstantigen is reduced by at least 30%, 40%, 50%, 60%, 70%, 80% or 90%relative to the maximal binding between the test antibody and the secondantigen, e.g., in a given experiment, or using mean values from multipleexperiments, as assessed by, e.g., a binding assay disclosed herein.

As used herein, the terms “specifically binds,” “specificallyrecognizes,” “immunospecifically binds,” and “immunospecificallyrecognizes” are analogous terms in the context of antibodies and referto molecules that bind to an antigen (e.g., epitope or immune complex)as such binding is understood by one skilled in the art. For example, amolecule that specifically binds to an antigen can bind to otherpeptides or polypeptides, generally with lower affinity as determinedby, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (SapidyneInstruments, Boise, ID), or other assays known in the art. In a specificembodiment, molecules that specifically bind to an antigen bind to theantigen with a K_(A) that is at least 2 logs (e.g., factors of 10), 2.5logs, 3 logs, 4 logs or greater than the K_(A) when the molecules bindnon-specifically to another antigen. The skilled worker will appreciatethat an antibody, as described herein, can specifically bind to morethan one antigen (e.g., via different regions of the antibody molecule).For example, an antibody, as described herein, can specifically bind toCD73 and TGFβ or specifically bind to CD73 and VEGF.

As used herein, the term “immunoglobulin chain” refers to a polypeptideof an antibody that comprises an antibody VH or VL, or anantigen-binding fragment thereof. An immunoglobulin chain can comprise,for example, a VH, a VL, a full-length antibody heavy chain, afull-length antibody light chain, or an antigen binding fragment of anyof the foregoing.

As used herein, the term “linked to” refers to covalent or noncovalentbinding between two molecules or moieties. The skilled worker willappreciate that when a first molecule or moiety is linked to a secondmolecule or moiety, the linkage need not be direct, but instead, can bevia an intervening molecule or moiety. For example, when a heavy chainvariable region of a full-length antibody is linked to a ligand-bindingmoiety, the ligand-binding moiety can bind a constant region (e.g., aheavy chain constant region) of the full-length antibody (e.g., via apeptide bond), rather than bind directly to the heavy chain variableregion.

In another specific embodiment, molecules that specifically bind to anantigen do not cross react with other proteins under similar bindingconditions. In another specific embodiment, molecules that specificallybind to CD73 do not cross react with other non-CD73 proteins. In aspecific embodiment, provided herein is an antibody that binds to CD73(e.g., human CD73) with higher affinity than to another unrelatedantigen. In certain embodiments, provided herein is an antibody thatbinds to CD73 (e.g., human CD73) with a 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinity thanto another, unrelated antigen as measured by, e.g., a radioimmunoassay,surface plasmon resonance, or kinetic exclusion assay. In a specificembodiment, the extent of binding of an anti-CD73 antibody describedherein to an unrelated, non-CD73 protein is less than 10%, 15%, or 20%of the binding of the antibody to CD73 protein as measured by, e.g., aradioimmunoassay.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody can specificallybind. An epitope can be, for example, contiguous amino acids of apolypeptide (linear or contiguous epitope) or an epitope can, forexample, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope to whichan antibody binds can be determined by, e.g., NMR spectroscopy, X-raydiffraction crystallography studies, ELISA assays, hydrogen/deuteriumexchange coupled with mass spectrometry (e.g., liquid chromatographyelectrospray mass spectrometry), array-based oligo-peptide scanningassays (e.g., constraining peptides using CLIPS (Chemical Linkage ofPeptides onto Scaffolds) to map discontinuous or conformationalepitopes), and/or mutagenesis mapping (e.g., site-directed mutagenesismapping). For X-ray crystallography, crystallization may be accomplishedusing any of the known methods in the art (e.g., Giegé R et al., (1994)Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A(1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274;McPherson A (1976) J Biol Chem 251: 6300-6303, each of which is hereinincorporated by reference in its entirety). Antibody:antigen crystalsmay be studied using well known X-ray diffraction techniques and may berefined using computer software such as X-PLOR (Yale University, 1992,distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol(1985) volumes 114 & 115, eds Wyckoff H W et al.; U.S. 2004/0014194),and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter C W;Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323), each of which is herein incorporated by reference in itsentirety. Mutagenesis mapping studies may be accomplished using anymethod known to one of skill in the art. See, e.g., Champe M et al.,(1995) J Biol Chem 270: 1388-1394 and Cunningham B C & Wells J A (1989)Science 244: 1081-1085, each of which is herein incorporated byreference in its entirety, for a description of mutagenesis techniques,including alanine scanning mutagenesis techniques. CLIPS (ChemicalLinkage of Peptides onto Scaffolds) is a technology to present one ormore peptides in a structurally constrained configuration to behave asfunctional mimics of complex protein domains. See, e.g., U.S.Publication Nos. US 2008/0139407 A1 and US 2007/099240 A1, and U.S. Pat.No. 7,972,993, each of which is herein incorporated by reference in itsentirety. In a specific embodiment, the epitope of an antibody isdetermined using alanine scanning mutagenesis studies. In a specificembodiment, the epitope of an antibody is determined usinghydrogen/deuterium exchange coupled with mass spectrometry. In aspecific embodiment, the epitope of an antibody is determined usingCLIPS Epitope Mapping Technology from Pepscan Therapeutics. In aspecific embodiment, the epitope of an antibody is determined by proteinmutagenesis, e.g., by generating switch mutants of an antigen withportions of its ortholog from another species and then testing theswitch mutants for loss of antibody binding (e.g., by a FACS-based cellbinding assay, as described herein).

As used herein, the term “an epitope located within” a region of humanCD73 refers to an epitope comprising one or more of the amino acidresidues of the specified region. In certain embodiments, the epitopecomprises each one of the amino acid residues located within thespecified region. In certain embodiments, the epitope consists of eachone of the amino acid residues located within the specified region. Incertain embodiments, one or more additional amino acid residues of humanCD73 outside the specified region bind to an antibody together with anepitope located within the specified region.

As used herein, the terms “T cell receptor” and “TCR” are usedinterchangeably and refer to full-length heterodimeric αβ or γδ TCRs,antigen-binding fragments of full-length TCRs, and molecules comprisingTCR CDRs or variable regions. Examples of TCRs include, but are notlimited to, full-length TCRs, antigen-binding fragments of full-lengthTCRs, soluble TCRs lacking transmembrane and cytoplasmic regions,single-chain TCRs containing variable regions of TCRs attached by aflexible linker, TCR chains linked by an engineered disulfide bond,monospecific TCRs, multi-specific TCRs (including bispecific TCRs), TCRfusions, human TCRs, humanized TCRs, chimeric TCRs, recombinantlyproduced TCRs, and synthetic TCRs. The term encompasses wild-type TCRsand genetically engineered TCRs (e.g., a chimeric TCR comprising achimeric TCR chain which includes a first portion from a TCR of a firstspecies and a second portion from a TCR of a second species).

As used herein, the terms “major histocompatibility complex” and “MHC”are used interchangeably and refer to an MHC class I molecule and/or anMHC class II molecule.

As used herein, the term “peptide-MHC complex” refers to an MHC molecule(MHC class I or MHC class II) with a peptide bound in the art-recognizedpeptide binding pocket of the MHC.

As used herein, the term “treat,” “treating,” and “treatment” refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration of an antibody to a subject having adisease or disorder, or predisposed to having such a disease ordisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disease or disorder or recurringdisease or disorder, or in order to prolong the survival of a subjectbeyond that expected in the absence of such treatment.

As used herein, the term “effective amount” in the context of theadministration of a therapy to a subject refers to the amount of atherapy that achieves a desired prophylactic or therapeutic effect.

As used herein, the term “subject” includes any human or non-humananimal. In one embodiment, the subject is a human or non-human mammal.In one embodiment, the subject is a human.

The determination of “percent identity” between two sequences (e.g.,amino acid sequences or nucleic acid sequences) can be accomplishedusing a mathematical algorithm. A specific, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin S & Altschul S F (1990) PNAS 87: 2264-2268,modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877, eachof which is herein incorporated by reference in its entirety. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul S F et al., (1990) J Mol Biol 215: 403, which is hereinincorporated by reference in its entirety. BLAST nucleotide searches canbe performed with the NBLAST nucleotide program parameters set, e.g.,for score=100, wordlength=12 to obtain nucleotide sequences homologousto a nucleic acid molecules described herein. BLAST protein searches canbe performed with the XBLAST program parameters set, e.g., to score 50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule described herein. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul S F etal., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated byreference in its entirety. Alternatively, PSI BLAST can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blastprograms, the default parameters of the respective programs (e.g., ofXBLAST and NBLAST) can be used (see, e.g., National Center forBiotechnology Information (NCBI) on the worldwide web,ncbi.nlm.nih.gov). Another specific, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is hereinincorporated by reference in its entirety. Such an algorithm isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

As used herein, the term “lipid nanoparticle” refers to one or morespherical nanoparticles with an average diameter of between about 10 toabout 1000 nanometers, and which comprise a solid lipid core matrix thatcan solubilize lipophilic molecules. In certain embodiments, the lipidcore is stabilized by surfactants (e.g., emulsifiers), and can compriseone or more of triglycerides (e.g., tristearin), diglycerides (e.g.,glycerol bahenate), monoglycerides (e.g., glycerol monostearate), fattyacids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes(e.g., cetyl palmitate), including combinations thereof. Lipidnanoparticles are described, for example, in Petrilli et al., Curr PharmBiotechnol. 15:847-55, 2014; and U.S. Pat. Nos. 6,217,912; 6,881,421;7,402,573; 7,404,969; 7,550,441; 7,727,969; 8,003,621; 8,691,750;8,871,509; 9,017,726; 9,173,853; 9,220,779; 9,227,917; and 9,278,130,each of which is incorporated by reference in its entirety.

5.2 Anti-CD73 Antibodies

In one aspect, the instant disclosure provides antibodies thatspecifically bind to CD73 (e.g., human CD73, cynomolgus CD73, or mouseCD73) and antagonize CD73 function. The amino acid sequences ofexemplary antibodies are set forth in Table 1, herein.

TABLE 1 Amino acid sequences of exemplary anti-CD73 antibodies. SEQDescription Amino Acid Sequence ID NO BA010 CDRH1 NYWMH   1 (Kabat)BA011 CDRH1 SSWIN   2 (Kabat) BA010 CDRH2 TIYPRNSDTNYNQKFKG   3 (Kabat)BA011 CDRH2 RIYPRNGDTNYNGKFKD   4 (Kabat) BA023 CDRH2 RIYPRAGDTNYAGKFKD  5 (Kabat) BA024 CDRH2 RIYPRSGDTNYSGKFKD   6 (Kabat) BA010 CDRH3LLDYSMDY   7 (Kabat) BA010 CDRL1 RASQDISNYLN   8 (Kabat) BA021 CDRL1RASQDISISLN   9 (Kabat) BA010 CDRL2 YTSRLHS  10 (Kabat) BA010 CDRL3QQGNTLPWT  11 (Kabat) BA012 CDRL3 QQGNTLPLT  12 (Kabat) BA025 CDRH1GYAFSSSW 143 (IMGT unique) BA025 CDRH2 IYPRAGDT 144 (IMGT unique)BA025 CDRH3 ASLLDYSMDY 145 (IMGT unique) BA025 CDRL1 QDISNY 146(IMGT unique) BA025 CDRL2 YTS 147 (IMGT unique) BA025 CDRL3 QQGNTLPLT 12 (IMGT unique) BA025 CDRH1 GYAFSSS 148 (Chothia) BA025 CDRH2 YPRAGD149 (Chothia) BA025 CDRH3 LLDYSMDY   7 (Chothia) BA025 CDRL1 RASQDISNYLN  8 (Chothia) BA025 CDRL2 YTSRLHS  10 (Chothia) BA025 CDRL3 QQGNTLPLT 12 (Chothia) BA025 CDRH1 ASGYAFSSSW 150 (AHo) BA025 CDRH2IYPRAGDTNYAGKFKDQ 151 (AHo) BA025 CDRH3 LLDYSMD 152 (AHo) BA025 CDRL1ASQDISNY 153 (AHo) BA025 CDRL2 YTSRLHSGVPSR 154 (AHo) BA025 CDRL3 GNTLPL155 (AHo) BA020 CDRH1 GYAFSSSW 143 (IMGT unique) BA020 CDRH2 IYPRNGDT156 (IMGT unique) BA020 CDRH3 ASLLDYSMDY 145 (IMGT unique) BA020 CDRL1QDISNY 146 (IMGT unique) BA020 CDRL2 YTS 147 (IMGT unique) BA020 CDRL3QQGNTLPLT  12 (IMGT unique) BA020 CDRH1 GYAFSSS 148 (Chothia)BA020 CDRH2 YPRNGD 157 (Chothia) BA020 CDRH3 LLDYSMDY   7 (Chothia)BA020 CDRL1 RASQDISNYLN   8 (Chothia) BA020 CDRL2 YTSRLHS  10 (Chothia)BA020 CDRL3 QQGNTLPLT  12 (Chothia) BA020 CDRH1 ASGYAFSSSW 150 (AHo)BA020 CDRH2 IYPRNGDTNYNGKFKDQ 158 (AHo) BA020 CDRH3 LLDYSMD 152 (AHo)BA020 CDRL1 ASQDISNY 153 (AHo) BA020 CDRL2 YTSRLHSGVPSR 154 (AHo)BA020 CDRL3 GNTLPL 155 (AHo) CDRH1 consensus X₁X₂WX₃X₄, wherein:  13sequence X₁ is S or N; X₂ is S or Y; X₃ is I or M; and X₄ is N or HCDRH2 consensus X₁IYPRX₂X₃DTNYX₄X₅KFKX₆, wherein:  14 sequence 1X₁ is R or T; X₂ is N, A, or S; X₃ is G or S; X₄ is N, A, or S;X₅ is G or Q; and X₆ is D or G CDRH2 consensusRIYPRX₁GDTNYX₂GKFKD, wherein:  15 sequence 2 X₁ is N, A, or S; andX₂ is N, A, or S CDRL1 consensus RASQDISX₁X₂LN, wherein:  16 sequenceX₁ is N or I; and X₂ is Y or S CDRL3 consensus QQGNTLPXT, wherein:  17sequence X is L or W Murine V_(H) germlineQVQLQQSGPELVKPGASVKISCKASGYTFTSYYIHWVK  18 sequenceQRPGQGLEWIGYIYPRDGSTNYNEKFKGKATLTADTSS muIgHV1S12 STAYMQLSSLTSEDSAVYFCARBA010 V_(H) (murine) XVQLQQPGTVLARPGASVKMSCKTSGYTFTNYWMEIW  19VKQRPGQGLEWIGTIYPRNSDTNYNQKFKGKAKLTAVTSASTAYMELSSLTNEDSAIYYCASLLDYSMDYWGQGTSVTVSS, wherein X is Q or pyroglutamate BA011 V_(H) (murine)XVQLKQSGPELVKPGASVKISCKASGYAFSSSWINWVN  20QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSSSTAYLQLSSLTPEDSAVYFCASLLDYSMDYWGQGTSVTVSS, wherein X is Q or pyroglutamate BA012 V_(H) (murine)XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  21QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSSSTAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTVSS, wherein X is E or pyroglutamate Human V_(H) germlineEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWTGWV  22 sequence huIgHV5-RQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSI 51*02 STAYLQWSSLKASDTAMYYCARBA019 V_(H) XVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWINWVR  23 (humanized)QMPGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARLLDYSMDYWGQGTLVTVSS, wherein X is E or pyroglutamate BA020 V_(H)XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVR  24 (humanized)QMPGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSS, wherein X is E or pyroglutamate BA023 V_(H)XVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWINWVR  25 (humanized)QMPGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARLLDYSMDYWGQGTLVTVSS, wherein X is E or pyroglutamate BA024 V_(H)XVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWINWVR  26 (humanized)QMPGKGLEWMGRIYPRSGDTNYSGKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARLLDYSMDYWGQGTLVTVSS, wherein X is E or pyroglutamate BA025 V_(H)XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVR  27 (humanized)QMPGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSS, wherein X is E or pyroglutamate BA026 V_(H)XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVR  28 (humanized)QMPGKGLEWMGRIYPRSGDTNYSGKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSS, wherein X is E or pyroglutamate BA031 V_(H)XVKLVESGAEVKKPGESLKISCKASGYAFSSSWINWVN  29 (humanized)QMPGKGLEWIGRIYPRNGDTNYNGKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSS, wherein X is E or pyroglutamate BA032 V_(H)XVKLVESGAEVKKPGESLKISCKASGYAFSSSWINWVN  30 (humanized)QMPGKGLEWIGRIYPRNGDTNYNGKFKDQVTLSADRSSSTAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSS, wherein X is E or pyroglutamate Humanized V_(H)X₁VQLVQSGAEVKKPGESLKISCKX₂SGYAFSSSWINWV  31 consensus sequenceRQMPGKGLEWMGRIYPRX₃GDTNYX₄GKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCAX₅LLDYSMDYWGQ GTLVTVSS, wherein:X₁ is E or pyroglutamate; X₂ is G or A; X₃ is N, A, or S;X₄ is N, A, or S; and X₅ is R or S Murine V_(L) germlineDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQ  32 sequence muIgKV10-KPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNL 96 EQEDIATYFCQQGNTLPBA010 V_(L) (murine) DIQMTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQK  33PDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLE QEDIATYFCQQGNTLPWTFGGGTKLEIKBA011 V_(L) (murine) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQ  34KPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNL EQEDIATYFCQQGNTLPWTFGGGTKLEIKBA012 V_(L) (murine) DIQMTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQK  35PDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLE QEDIATYFCQQGNTLPLTFGAGTKLELKHuman V_(L) germline DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQ  36sequence huIgKV1- KPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSL 33*01QPEDIATYYCQQYDNLPP BA019 V_(L) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQ 37 (humanized) KPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPLTFGQGTKVEIK BA021 V_(L)DIQMTQSPSSLSASVGDRVTITCRASQDISISLNWYQQK  38 (humanized)PGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQ PEDIATYYCQQGNTLPLTFGQGTKVEIKBA033 V_(L) DIQMTQTPSSLSASVGDRVTISCRASQDISISLNWYQQK  39 (humanized)PGKTPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQ PEDIATYYCQQGNTLPLTFGQGTKVEIKBA034 V_(L) DIQMTQTTSSLSASVGDRVTISCRASQDISISLNWYQQK  40 (humanized)PGKTPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQ PEDIATYFCQQGNTLPLTFGQGTKVEIKHumanized V_(L) DIQMTQSPSSLSASVGDRVTITCRASQDISX₁X₂LNWYQ  41consensus sequence 1 QKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDX₃TFTISSLQPEDIATYYCQQGNTLPLTFGQGTKVEIK, wherein: X₁ is N or I;X₂ is Y or S; and X₃ is For Y Humanized V_(L)DIQMTQSPSSLSASVGDRVTITCRASQDISX₁X₂LNWYQ  42 consensus sequence 2QKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDX₃TFTISSLQPEDIATYYCQQGNTLPLTFGQGTKVEIK, wherein: X₁X₂ is NY or IS; andX₃ is For Y Mouse IgG2a heavy AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVT  43chain constant region LTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS CSVVHEGLHNHEITTKSFSRTPGKMouse IgG2a heavy AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVT  44chain constant region LTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS(without C-terminal QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNL lysine)LGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS CSVVHEGLHNHHTTKSFSRTPGMouse IgG2a heavy AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVT  45chain constant region LTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSwith N297A mutation QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS CSVVHEGLHNHHTTKSFSRTPGKMouse IgG2a heavy AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVT  46chain constant region LTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSwith N297A mutation QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNL(without C-terminal LGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQ lysine)ISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS CSVVHEGLHNHHTTKSFSRTPGHuman IgG₁ heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV  47chain constant region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG₁ heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV  48chain constant region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT(without C-terminal QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE lysine)LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGHuman IgG₁ heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV  49chain constant region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTwith N297A mutation QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG₁ heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV  50chain constant region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTwith N297A mutation QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE(without C-terminal LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE lysine)VKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGHuman IgG₂ heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV  51chain constant region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKHuman IgG₂ heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV  52chain constant region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT(without C-terminal QTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA lysine)GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGHuman IgG₁ A330S- ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV  53P331S/G₂ hybrid SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTheavy chain constant QTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAG regionPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKHuman IgG₁ A330S- ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV  54P331S/G₂ hybrid SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTheavy chain constant QTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGregion (without C- PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNterminal lysine) WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG Human IgG₁ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV  55 N297A/G₂ hybridSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT heavy chain constantQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAG regionPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK Human IgG₁ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV  56 N297A/G₂ hybridSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT heavy chain constantQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAG region (without C-PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN terminal lysine)WYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGHuman IgG₄ heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV  57chain constant region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTwith S228P mutation KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGKHuman IgG₄ heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV  58chain constant region SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTwith S228P mutation KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG(without C-terminal GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF lysine)NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGB A012 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  63murine heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS (murine IgG2aSTAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVT backbone withVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEP N297A mutation)VTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK, wherein X is E or pyroglutamateBA012 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  64murine heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS (murine IgG2aSTAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVT backbone withVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEP N297A mutation andVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSST without C-terminalWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPA lysine)PNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG, wherein X is E or pyroglutamateBA013 full-length XVQLQQPGTVLARPGASVKMSCKTSGYTFTNYWMHW  65chimeric heavy chain VKQRPGQGLEWIGTIYPRNSDTNYNQKFKGKAKLTAV(murine variable TSASTAYMELSSLTNEDSAIYYCASLLDYSMDYWGQGdomain + human IgG1 TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYconstant domain with FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVN297A mutation) PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is Q or pyroglutamateBA013 full-length XVQLQQPGTVLARPGASVKMSCKTSGYTFTNYWMHW  66chimeric heavy chain VKQRPGQGLEWIGTIYPRNSDTNYNQKFKGKAKLTAV(murine variable TSASTAYMELSSLTNEDSAIYYCASLLDYSMDYWGQGdomain + human IgG1 TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYconstant domain with FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVN297A mutation and PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCwithout C-terminal PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD lysine)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is Q or pyroglutamateBA014 full-length XVQLKQSGPELVKPGASVKISCKASGYAFSSSWINWVN  67chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTPEDSAVYFCASLLDYSMDYWGQGTSVTdomain + human IgG1 VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPconstant domain with VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSN297A mutation) LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is Q or pyroglutamateBA014 full-length XVQLKQSGPELVKPGASVKISCKASGYAFSSSWINWVN  68chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTPEDSAVYFCASLLDYSMDYWGQGTSVTdomain + human IgG1 VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPconstant domain with VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSN297A mutation and LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPwithout C-terminal APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE lysine)DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is Q or pyroglutamateBA015 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  69chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTdomain + human IgG1 VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPconstant domain with VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSN297A mutation) LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA015 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  70chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTdomain + human IgG1 VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPconstant domain with VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSN297A mutation and LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPwithout C-terminal APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE lysine)DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateBA016 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  71chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTregion + human IgG₂ VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPconstant region) VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA016 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  72chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTregion + human IgG₂ VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPconstant region and VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSwithout C-terminal NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAP lysine)PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateBA017 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  73chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTregion + human IgG₁- VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPA330S-P331S/IgG₂ VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS hybrid constantNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAP region)PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA017 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  74chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTregion + human IgG₁- VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPA330S-P331S/IgG₂ VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS hybrid constantNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAP region and without C-PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE terminal lysine)VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateBA018 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  75chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTregion + human IgG₁- VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPN297A/IgG₂ hybrid VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSconstant region) NFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA018 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVN  76chimeric heavy chain QRPGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSS(murine variable STAYLQLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTregion + human IgG₁- VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPN297A/IgG₂ hybrid VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSconstant region and NFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPwithout C-terminal PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE lysine)VKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateBA019 full-length XVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWINWVR  77humanized heavy QMPGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKS chain (human IgG₁ISTAYLQWSSLKASDTAMYYCARLLDYSMDYWGQGT backbone withLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF N297A mutation)PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA019 full-length XVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWINWVR  78humanized heavy QMPGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKS chain (human IgG₁ISTAYLQWSSLKASDTAMYYCARLLDYSMDYWGQGT backbone withLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF N297A mutation andPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP without C-terminalSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP lysine)PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateBA020 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVR 79humanized heavy QMPGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKS chain (human IgG₁ISTAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTL backbone withVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP N297A mutation)EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA020 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVR  80humanized heavy QMPGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKS chain (human IgG₁ISTAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTL backbone withVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP N297A mutation andEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS without C-terminalSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP lysine)CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateBA023 full-length XVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWINWVR  81humanized heavy QMPGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKS chain (human IgG1ISTAYLQWSSLKASDTAMYYCARLLDYSMDYWGQGT backbone withLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF N297A mutation)PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA023 full-length XVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWINWVR  82humanized heavy QMPGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKS chain (human IgG₁ISTAYLQWSSLKASDTAMYYCARLLDYSMDYWGQGT backbone withLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF N297A mutation andPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP without C-terminalSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP lysine)PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateBA024 full-length XVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWINWVR  83humanized heavy QMPGKGLEWMGRIYPRSGDTNYSGKFKDQVTISADKSI chain (human IgG₁STAYLQWSSLKASDTAMYYCARLLDYSMDYWGQGTL backbone withVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP N297A mutation)EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA024 full-length XVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWINWVR  84humanized heavy QMPGKGLEWMGRIYPRSGDTNYSGKFKDQVTISADKSI chain (human IgG₁STAYLQWSSLKASDTAMYYCARLLDYSMDYWGQGTL backbone withVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP N297A mutation andEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS without C-terminalSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP lysine)CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateBA025 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVR  85humanized heavy QMPGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKS chain (human IgG₁ISTAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTL backbone withVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP N297A mutation)EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA025 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVR  86humanized heavy QMPGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKS chain (human IgG₁ISTAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTL backbone withVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP N297A mutation andEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS without C-terminalSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP lysine)CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateBA026 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVR  87humanized heavy QMPGKGLEWMGRIYPRSGDTNYSGKFKDQVTISADKSI chain (human IgG₁STAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTL backbone withVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP N297A mutation)EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, wherein X is E or pyroglutamateBA026 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVR  88humanized heavy QMPGKGLEWMGRIYPRSGDTNYSGKFKDQVTISADKSI chain (human IgG₁STAYLQWSSLKASDTAMYYCASLLDYSMDYWGQGTL backbone withVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP N297A mutation andEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS without C-terminalSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP lysine)CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, wherein X is E or pyroglutamateMurine Igκ light RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV  89chain constant region KWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC BA012 full-lengthDIQMTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQK  92 light chain (murine)PDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYT CEATHKTSTSPIVKSFNRNECHuman Igic light RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV  93chain constant region QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC BA013 full-lengthDIQMTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQK  94 light chain (chimeric;PDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLE murine variable +QEDIATYFCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIF human Igκ constantPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ region)SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECBA014 full-length DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQ  95light chain (chimeric; KPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLmurine variable + EQEDIATYFCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIhuman Igκ constant FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL region)QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECBA015 full-length DIQMTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQK  96light chain (chimeric; PDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEmurine variable + QEDIATYFCQQGNTLPLTFGAGTKLELKRTVAAPSVFIFhuman Igκ constant PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ region)SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECBA019 full-length DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQ  97 light chainKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSL (humanized)QPEDIATYYCQQGNTLPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECBA021 full-length DIQMTQSPSSLSASVGDRVTITCRASQDISISLNWYQQK  98light chain PGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQ (humanized)PEDIATYYCQQGNTLPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC

TABLE 2 Heavy chain CDR sequences of exemplary anti-CD73 antibodiesCDRH1 Anti- (SEQ CDRH2 (SEQ CDRH3 (SEQ body ID NO) ID NO) ID NO) BA010NYWMH (1) TIYPRNSDTNYNQKFKG (3) LLDYSMDY (7) BA011 SSWIN (2)RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA012 SSWIN (2) RIYPRNGDTNYNGKFKD (4)LLDYSMDY (7) BA013 NYWMH (1) TIYPRNSDTNYNQKFKG (3) LLDYSMDY (7) BA014SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA015 SSWIN (2)RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA016 SSWIN (2) RIYPRNGDTNYNGKFKD (4)LLDYSMDY (7) BA017 SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA018SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA019 SSWIN (2)RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA020 SSWIN (2) RIYPRNGDTNYNGKFKD (4)LLDYSMDY (7) BA021 SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA022SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA023 SSWIN (2)RIYPRAGDTNYAGKFKD (5) LLDYSMDY (7) BA024 SSWIN (2) RIYPRSGDTNYSGKFKD (6)LLDYSMDY (7) BA025 SSWIN (2) RIYPRAGDTNYAGKFKD (5) LLDYSMDY (7) BA026SSWIN (2) RIYPRSGDTNYSGKFKD (6) LLDYSMDY (7) BA027 SSWIN (2)RIYPRAGDTNYAGKFKD (5) LLDYSMDY (7) BA028 SSWIN (2) RIYPRSGDTNYSGKFKD (6)LLDYSMDY (7) BA029 SSWIN (2) RIYPRAGDTNYAGKFKD (5) LLDYSMDY (7) BA030SSWIN (2) RIYPRSGDTNYSGKFKD (6) LLDYSMDY (7) BA031 SSWIN (2)RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA032 SSWIN (2) RIYPRNGDTNYNGKFKD (4)LLDYSMDY (7) BA033 SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA034SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA035 SSWIN (2)RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA036 SSWIN (2) RIYPRNGDTNYNGKFKD (4)LLDYSMDY (7) BA037 SSWIN (2) RIYPRAGDTNYAGKFKD (5) LLDYSMDY (7) BA038SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA039 SSWIN (2)RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA040 SSWIN (2) RIYPRAGDTNYAGKFKD (5)LLDYSMDY (7) BA041 SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA042SSWIN (2) RIYPRAGDTNYAGKFKD (5) LLDYSMDY (7) BA043 SSWIN (2)RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA044 SSWIN (2) RIYPRAGDTNYAGKFKD (5)LLDYSMDY (7) BA045 SSWIN (2) RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA046SSWIN (2) RIYPRAGDTNYAGKFKD (5) LLDYSMDY (7) BA047 SSWIN (2)RIYPRNGDTNYNGKFKD (4) LLDYSMDY (7) BA048 SSWIN (2) RIYPRAGDTNYAGKFKD (5)LLDYSMDY (7) The V_(H) CDRs in Table 2 are determined according toKabat.

The V_(H) CDRs in Table 2 are determined according to Kabat.

TABLE 3 Light chain CDR sequences of exemplary anti-CD73  antibodiesAnti- CDRL1 (SEQ CDRL2 (SEQ CDRL3 (SEQ body ID NO) ID NO) ID NO) BA010RASQDISNYLN (8) YTSRLHS (10) QQGNTLPWT (11) BA011 RASQDISNYLN (8)YTSRLHS (10) QQGNTLPWT (11) BA012 RASQDISNYLN (8) YTSRLHS (10)QQGNTLPLT (12) BA013 RASQDISNYLN (8) YTSRLHS (10) QQGNTLPWT (11) BA014RASQDISNYLN (8) YTSRLHS (10) QQGNTLPWT (11) BA015 RASQDISNYLN (8)YTSRLHS (10) QQGNTLPLT (12) BA016 RASQDISNYLN (8) YTSRLHS (10)QQGNTLPLT (12) BA017 RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA018RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA019 RASQDISNYLN (8)YTSRLHS (10) QQGNTLPLT (12) BA020 RASQDISNYLN (8) YTSRLHS (10)QQGNTLPLT (12) BA021 RASQDISISLN (9) YTSRLHS (10) QQGNTLPLT (12) BA022RASQDISISLN (9) YTSRLHS (10) QQGNTLPLT (12) BA023 RASQDISNYLN (8)YTSRLHS (10) QQGNTLPLT (12) BA024 RASQDISNYLN (8) YTSRLHS (10)QQGNTLPLT (12) BA025 RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA026RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA027 RASQDISISLN (9)YTSRLHS (10) QQGNTLPLT (12) BA028 RASQDISISLN (9) YTSRLHS (10)QQGNTLPLT (12) BA029 RASQDISISLN (9) YTSRLHS (10) QQGNTLPLT (12) BA030RASQDISISLN (9) YTSRLHS (10) QQGNTLPLT (12) BA031 RASQDISISLN (9)YTSRLHS (10) QQGNTLPLT (12) BA032 RASQDISISLN (9) YTSRLHS (10)QQGNTLPLT (12) BA033 RASQDISISLN (9) YTSRLHS (10) QQGNTLPLT (12) BA034RASQDISISLN (9) YTSRLHS (10) QQGNTLPLT (12) BA035 RASQDISNYLN (8)YTSRLHS (10) QQGNTLPLT (12) BA036 RASQDISNYLN (8) YTSRLHS (10)QQGNTLPLT (12) BA037 RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA038RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA039 RASQDISNYLN (8)YTSRLHS (10) QQGNTLPLT (12) BA040 RASQDISNYLN (8) YTSRLHS (10)QQGNTLPLT (12) BA041 RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA042RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA043 RASQDISNYLN (8)YTSRLHS (10) QQGNTLPLT (12) BA044 RASQDISNYLN (8) YTSRLHS (10)QQGNTLPLT (12) BA045 RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA046RASQDISNYLN (8) YTSRLHS (10) QQGNTLPLT (12) BA047 RASQDISNYLN (8)YTSRLHS (10) QQGNTLPLT (12) BA048 RASQDISNYLN (8) YTSRLHS (10)QQGNTLPLT (12) The V_(L) CDRs in Table 3 are determined according toKabat.

The V_(L) CDRs in Table 3 are determined according to Kabat.

TABLE 4 VH, VL, full-length heavy chain, and full-length light chainsequences of exemplary anti-CD73 antibodies* Full-length Full-lengthV_(H) heavy chain V_(L) light chain Antibody SEQ ID NO. SEQ ID NO. SEQID NO. SEQ ID NO. BA010 19 N/A 33 N/A BA011 20 N/A 34 N/A BA012 21 64 3592 BA013 19 65 33 94 BA014 20 67 34 95 BA015 21 70 35 96 BA016 21 71 3596 BA017 21 73 35 96 BA018 21 75 35 96 BA019 23 77 37 97 BA020 24 79 3797 BA021 23 77 38 98 BA022 24 79 38 98 BA023 25 81 37 97 BA024 26 83 3797 BA025 27 85 37 97 BA026 28 87 37 97 BA027 25 81 38 98 BA028 26 83 3898 BA029 27 85 38 98 BA030 28 87 38 98 BA031 29 N/A 37 N/A BA032 30 N/A37 N/A BA033 23 N/A 39 N/A BA034 23 N/A 40 N/A BA035 21 113 35 92 BA03624 114 37 97 BA037 27 115 37 97 BA038 21 123 35 92 BA039 24 124 37 128BA040 27 125 37 128 BA041 24 126 37 97 BA042 27 127 37 97 BA043 24 11637 97 BA044 27 117 37 97 BA045 24 118 37 97 BA046 27 119 37 97 BA047 24120 37 97 BA048 27 121 37 97 *In this table, a polypeptide of ananti-CD73 antibody comprising a ligand binding moiety is referred to asa full-length heavy chain if it comprises a full-length heavy chain.

TABLE 5 Exemplary sequences of CD73 and family members. SEQ DescriptionAmino Acid Sequence ID NO Human CD73MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSR  99 isoform 1LEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLL (NCBIDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEF ReferenceDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKV Sequence:LPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDK NP_002517.1)LKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSLIFLSLWAVIFVLYQ Human CD73MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSR 100 isoform 2LEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLL (NCBIDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEF ReferenceDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKV Sequence:LPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDK NP_001191742.1)LKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFST GSHCHGSFSLIFLSLWAVIFVLYQHuman CD73 MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSR 129 isoform 3LEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLL (NCBIDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEF ReferenceDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKV Sequence:LPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDK AAH65937)LKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHADETFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSLIFLSLWAVIFVLYQ Human CD73MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSR 130 isoform 4LEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLL (NCBIDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEF ReferenceDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKV Sequence:LPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDK EAW48635.1)LKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHADEMFWNHVSMCILNGGGIRSPIDERNNGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKF STGSHCHGSFSLIFLSLWAVIFVLYQCynomolgus MCPRAARAPATLLLAVGALLWSAAGAWELTILHTNDVHSR 101 CD73 variant 1LEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLL (NCBIDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEF ReferenceDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKV Sequence:LPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDK XP_001086989.1)LKTLNVNKIIALGHSGFETDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHADEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEIYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSLIFLSFCAVIFVLYQ CynomolgusMCPRAARAPAKLLLAVGALLWSAAGAWELTILHTNDVHSR 131 CD73 variant 2LEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLL (NCBIDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEF ReferenceDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKV Sequence:LPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDK EHH53214.1)LKTLNVNKIIALGHSGFETDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHADEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEIYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSLIFLSFCAVIFVLYQ Mouse CD73MRPAAAKVPKWLLLALSALLPQWPAASAWELTILHTNDVH 102 (NCBISRLEQTSDDSTKCLNASLCVGGVARLFTKVQQIRKEEPNVLF ReferenceLDAGDQYQGTIWFTVYKGLEVAHFMNILGYDAMALGNHEF Sequence:DNGVEGLIDPLLRNVKFPILSANIKARGPLAHQISGLFLPSKV NP_035981.1)LSVGGEVVGIVGYTSKETPFLSNPGTNLVFEDEISALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDIVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTADDGRQVPVVQAYAFGKYLGYLKVEFDDKGNVITSYGNPILLNSSIPEDATIKADINQWRIKLDNYSTQELGRTIVYLDGSTQTCRFRECNIVIGNLICDAMINNNLRHPDEMFWNHVSMCIVNGGGIRSPIDEKNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDINRKPWNRVVQLEVLCTKCRVPIYEPLEMDKVYKVTLPSYLANGGDGFQMIKDELLKHDSGDQDISVVSEYISKMKVVYPAVEGRIKFSAASHYQGSFPLVILSFWAMILILYQ Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK 141 isoform 1 withVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA His-tagLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHHHG GSGGLPETGGDR Mouse CD73WELTILHTNDVHSRLEQTSDDSTKCLNASLCVGGVARLFTK 142 with His-tagVQQIRKEEPNVLFLDAGDQYQGTIWFTVYKGLEVAHFMNILGYDAMALGNHEFDNGVEGLIDPLLRNVKFPILSANIKARGPLAHQISGLFLPSKVLSVGGEVVGIVGYTSKETPFLSNPGTNLVFEDEISALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDIVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTADDGRQVPVVQAYAFGKYLGYLKVEFDDKGNVITSYGNPILLNSSIPEDATIKADINQWRIKLDNYSTQELGRTIVYLDGSTQTCRFRECNMGNLICDAMINNNLRHPDEMFWNHVSMCIVNGGGIRSPIDEKNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDINRKPWNRVVQLEVLCTKCRVPIYEPLEMDKVYKVTLPSYLANGGDGFQMIKDELLKHDSGDQDISVVSEYISKMKVVYPAVEGRIKENLYFQGLEHHHHHHHHHH GGSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK  59 Y158A mutantVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA with His-tagLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLALPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNNIGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHHHG GSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK  60 Y161A mutantVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA with His-tagLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPAKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNNIGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHHHG GSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK 139 P165A mutantVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA with His-tagLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLAVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHH HGGSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK 140 D168A mutantVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA with His-tagLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGAEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNNIGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHHHG GSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK  61 T198A mutantVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA with His-tagLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEIAALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNNIGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHHHG GSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK  62 K274A mutantVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA with His-tagLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRAVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNNIGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHHHG GSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK 132 S269A mutantVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA with His-tagLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTADDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHHHG GSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK 133 I158A,VQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA Y161A, P165ALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGP D168A mutantLASQISGLALPAKVLAVGAEVVGIVGYTSKETPFLSNPGTNL with His-tagVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHH HGGSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK 134 Y158F, Y161S,VQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA P165S, D168GLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGP mutant withLASQISGLFLPSKVLSVGGEVVGIVGYTSKETPFLSNPGTNLV His-tagFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHHHG GSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK 135 S152H, Y158F,VQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA Y1615 mutantLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGP with His-tagLAHQISGLFLPSKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHHHG GSGGLPETGGDR Human CD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTK 136 P165S, D168GVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNA mutant withLRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGP His-tagLASQISGLYLPYKVLSVGGEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKENLYFQGLEHHHHHHHHH HGGSGGLPETGGDR Mouse CD73WELTILHTNDVHSRLEQTSDDSTKCLNASLCVGGVARLFTK 137 I109A, G111R,VQQIRKEEPNVLFLDAGDQYQGTIWFTVYKGLEVAHFMNAL H154S, F160Y,RYDAMALGNHEFDNGVEGLIDPLLRNVKFPILSANIKARGPL S163Y, S167P,ASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVF G170D, S200T,EDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRG D298E, K299RVDIVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTADDGRQVPV mutant withVQAYAFGKYLGYLKVEFDERGNVITSYGNPILLNSSIPEDATI His-tagKADINQWRIKLDNYSTQELGRTIVYLDGSTQTCRFRECNMGNLICDAMINNNLRHPDEMFWNHVSMCIVNGGGIRSPIDEKNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDINRKPWNRVVQLEVLCTKCRVPIYEPLEMDKVYKVTLPSYLANGGDGFQMIKDELLKHDSGDQDISVVSEYISKMKVVYPAVEGRIKENLYFQGLEHHHHHHHHHH GGSGGLPETGGDR Mouse CD73WELTILHTNDVHSRLEQTSDDSTKCLNASLCVGGVARLFTK 138 S163Y, S167P,VQQIRKEEPNVLFLDAGDQYQGTIWFTVYKGLEVAHFMNIL G170D mutantGYDAMALGNHEFDNGVEGLIDPLLRNVKFPILSANIKARGPL with His-tagAHQISGLFLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEISALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDIVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTADDGRQVPVVQAYAFGKYLGYLKVEFDDKGNVITSYGNPILLNSSIPEDATIKADINQWRIKLDNYSTQELGRTIVYLDGSTQTCRFRECNMGNLICDAMINNNLRHPDEMEWNHVSMCIVNGGGIRSPIDEKNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDINRKPWNRVVQLEVLCTKCRVPIYEPLEMDKVYKVTLPSYLANGGDGFQMIKDELLKHDSGDQDISVVSEYISKMKVVYPAVEGRIKENLYFQGLEHHHHHHHHHH GGSGGLPETGGDR

TABLE 6Exemplary sequences of linkers, ligand binding moieties, and anti-CD73 antibodiescomprising ligand binding moieties* SEQ ID DescriptionAmino Acid Sequence NO Peptide linker 1 GGGGSGGGGSGGGGSGGGGSG 103Peptide linker 2 GGGGSGGGGS 104 Peptide linker 3 GGGGSGGGGSGGGGS 105Peptide linker 4 GGGGSG 106 Peptide linker 5 GSAGSAAGSGEF 107Peptide linker 6 AEAAAKEAAAKA 108 TGFPR2IPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTD 109 extracellularNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE domain variant 1VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIM from TGFβR2KEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD isoform A TGFPR2TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVT 110 extracellularDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP domain variant 2QEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKC from TGFβR2IMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLL isoform A VIFQ TGFPR2IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQ 111 extracellularKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL domain variant 3PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI from TGFβR2 IFSEEYNTSNPDisoform B TGFPR2 TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDN 112extracellular QKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDP domain variant 4KLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECND from TGFβR2NIIFSEEYNTSNPDLLLVIFQ isoform B BA035 full-lengthXVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVNQRP 113 murine heavyGKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSSSTAYLQ chainLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCS SDECNDNIIFSEEYNTSNPD,wherein X is E or pyroglutamate BA036 full-lengthXVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 114 humanized heavyPGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKSISTAYL chainQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD, wherein X is E or pyroglutamateBA037 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 115humanized heavy PGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKSISTAYL chainQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD, wherein X is E or pyroglutamateBA043 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 116humanized heavy PGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKSISTAYL chainQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD, wherein X is E or pyroglutamate BA044 full-lengthXVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 117 humanized heavyPGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKSISTAYL chainQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD, wherein X is E or pyroglutamate BA045 full-lengthXVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 118 humanized heavyPGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKSISTAYL chainQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAEAAAKEAAAKAIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD, wherein X is E or pyroglutamateBA046 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 119humanized heavy PGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKSISTAYL chainQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAEAAAKEAAAKAIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD, wherein X is E or pyroglutamateBA047 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 120humanized heavy PGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKSISTAYL chainQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSAGSAAGSGEFIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD, wherein X is E or pyroglutamateBA048 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 121humanized heavy PGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKSISTAYL chainQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSAGSAAGSGEFIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD, wherein X is E or pyroglutamate Human VEGFR1SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKF 122 extracellularPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHL domain YKTNYLTHRQTNTBA038 full-length XVKLVESGPELVKPGASVKISCKASGYAFSSSWINWVNQRP 123heavy chain GKGLEWIGRIYPRNGDTNYNGKFKDRATLTADRSSSTAYLQ (murine antibodyLSSLTSEDSAVYFCASLLDYSMDYWGQGTSVTVSSAKTTAP heavy chain +SVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLS human VEGFR1SGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPAS extracellularSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDV domain)LMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT, wherein X is E or pyroglutamate BA039 full-lengthXVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 124 heavy chain 1PGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKSISTAYL (humanizedQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSAKT variable + murineTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSG constant + humanSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAH VEGFR1PASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKI extracellularKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQ domain)TQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSSDTGRPFVEMYSEIPEIIRMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT, wherein X is E or pyroglutamate BA040 full-lengthXVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 125 heavy chain 2PGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKSISTAYL (humanizedQWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSAKT variable + murineTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSG constant + humanSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAH VEGFR1PASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKI extracellularKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQ domain)TQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGGGGGSGGGGSSDTGRPFVEMYSEIPEIIRMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT, wherein X is E or pyroglutamate BA041 full-lengthXVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 126 heavy chainPGKGLEWMGRIYPRNGDTNYNGKFKDQVTISADKSISTAYL (humanized)QWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT, wherein X is E or pyroglutamateBA042 full-length XVQLVQSGAEVKKPGESLKISCKASGYAFSSSWINWVRQM 127heavy chain PGKGLEWMGRIYPRAGDTNYAGKFKDQVTISADKSISTAYL (humanized)QWSSLKASDTAMYYCASLLDYSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT, wherein X is E or pyroglutamateBA039 full-length DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPG 128light chain KAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIAT (chimeric;YYCQQGNTLPLTFGQGTKVEIKRADAAPTVSIFPPSSEQLTS humanizedGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQD variable + murineSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF constant) NRNEC *In thistable, a polypeptide of an anti-CD73 antibody comprising a ligandbinding moiety is referred to as a full-length heavy chain if itcomprises a full-length heavy chain.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising a VH domain comprising one,two, or all three of the CDRs of a VH domain set forth in Table 1herein. In certain embodiments, the antibody comprises the CDRH1 of a VHdomain set forth in Table 1. In certain embodiments, the antibodycomprises the CDRH2 of a VH domain set forth in Table 1. In certainembodiments, the antibody comprises the CDRH3 of a VH domain set forthin Table 1.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising a VL domain comprising one,two, or all three of the CDRs of a VL domain disclosed in Table 1herein. In certain embodiments, the antibody comprises the CDRL1 of a VLdomain set forth in Table 1. In certain embodiments, the antibodycomprises the CDRL2 of a VL domain set forth in Table 1. In certainembodiments, the antibody comprises the CDRL3 of a VL domain set forthin Table 1.

In certain embodiments, the CDRs of an antibody can be determinedaccording to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) andKabat et al., Sequences of protein of immunological interest (1991),each of which is herein incorporated by reference in its entirety. Incertain embodiments, the light chain CDRs of an antibody are determinedaccording to Kabat and the heavy chain CDRs of an antibody aredetermined according to MacCallum (supra). In certain embodiments, heavychain CDRs and/or light chain CDRs are defined by performing structuralanalysis of an antibody and identifying residues in the variableregion(s) predicted to make contact with an epitope region of a targetmolecule (e.g., human and/or cynomolgus CD73).

In certain embodiments, the CDRs of an antibody can be determinedaccording to the Chothia numbering scheme, which refers to the locationof immunoglobulin structural loops (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917; A1-Lazikani B et al., (1997) J Mol Biol273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817;Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No.7,709,226, all of which are herein incorporated by reference in theirentireties). Typically, when using the Kabat numbering convention, theChothia CDRH1 loop is present at heavy chain amino acids 26 to 32, 33,or 34, the Chothia CDRH2 loop is present at heavy chain amino acids 52to 56, and the Chothia CDRH3 loop is present at heavy chain amino acids95 to 102, while the Chothia CDRL1 loop is present at light chain aminoacids 24 to 34, the Chothia CDRL2 loop is present at light chain aminoacids 50 to 56, and the Chothia CDRL3 loop is present at light chainamino acids 89 to 97. The end of the Chothia CDRH1 loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34).

In certain embodiments, the CDRs of an antibody can be determinedaccording to MacCallum R M et al., (1996) J Mol Biol 262: 732-745,herein incorporated by reference in its entirety. See also, e.g., MartinA. “Protein Sequence and Structure Analysis of Antibody VariableDomains,” in Antibody Engineering, Kontermann and Dubel, eds., Chapter31, pp. 422-439, Springer-Verlag, Berlin (2001), herein incorporated byreference in its entirety.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising the Chothia VH CDRs of a VHdisclosed in Table 1 herein. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), the antibody comprising theChothia VL CDRs of a VL disclosed in Table 1 herein. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73), theantibody comprising the Chothia VH CDRs and Chothia VL CDRs of anantibody disclosed in Table 1 herein. In certain embodiments, antibodiesthat specifically bind to CD73 (e.g., human, mouse, or cynomolgus CD73)comprise one or more CDRs, in which the Chothia and Kabat CDRs have thesame amino acid sequence. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD73 (e.g.,human, mouse, or cynomolgus CD73) and comprises combinations of KabatCDRs and Chothia CDRs.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the IMGT numbering system as described in Lefranc M-P,(1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999)Nucleic Acids Res 27: 209-212, each of which is herein incorporated byreference in its entirety. According to the IMGT numbering scheme, CDRH1is at positions 26 to 35, CDRH2 is at positions 51 to 57, CDRH3 is atpositions 93 to 102, CDRL1 is at positions 27 to 32, CDRL2 is atpositions 50 to 52, and CDRL3 is at positions 89 to 97. IMGT uniquenumbering may also be defined as in Lefranc M-P et al., (2009) NucleicAcids Res 37: D1006-D1012 and the IMGT database.

In certain embodiments, the instant disclosure provides antibodies thatspecifically bind to CD73 (e.g., human, mouse, or cynomolgus CD73) andcomprise CDRs of an antibody disclosed in Table 1 herein, as determinedby the IMGT numbering system, for example, as described in Lefranc M-P(1999) supra and Lefranc M-P et al., (1999) supra or Lefranc M-P et al.,(2009) supra.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the AbM numbering scheme, which refers to AbM hypervariableregions, which represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software (Oxford Molecular Group, Inc.), herein incorporated byreference in its entirety. In a particular embodiment, the instantdisclosure provides antibodies that specifically bind to CD73 (e.g.,human, mouse, or cynomolgus CD73) and comprise CDRs of an antibodydisclosed in Table 1 herein as determined by the AbM numbering scheme.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the AHo numbering system (as described in Honegger andPluckthun, A., J. Mol. Biol. 309:657-670 (2001), herein incorporated byreference in its entirety). In certain embodiments, the instantdisclosure provides antibodies that specifically bind to CD73 (e.g.,human, mouse, or cynomolgus CD73) and comprise CDRs of an antibodydisclosed in Table 1 herein as determined by the AHo numbering scheme.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), wherein the antibody comprises a heavy chain variableregion comprising the CDRH1, CDRH2, and CDRH3 region amino acidsequences of a VH domain set forth in SEQ ID NO: 19, 20, 21, 23, 24, 25,26, 27, 28, 29, or 30, and a light chain variable region comprising theCDRL1, CDRL2, and CDRL3 region amino acid sequences of a VL domain setforth in SEQ ID NO: 33, 34, 35, 37, 38, 39, or 40, wherein each CDR isdefined in accordance with the MacCallum definition, the Kabatdefinition, the Chothia definition, the IMGT numbering system, the AbMdefinition of CDR, structural analysis, or a combination thereof,wherein the structural analysis identifies residues in the variableregion(s) predicted to make contact with an epitope region of CD73(e.g., human, mouse, or cynomolgus CD73). In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), wherein the antibodycomprises a heavy chain variable region comprising the CDRH1, CDRH2, andCDRH3 region amino acid sequences of a VH domain, and a light chainvariable region comprising the CDRL1, CDRL2, and CDRL3 region amino acidsequences of a VL domain, wherein the VH domain and the VL domaincomprise the amino acid sequences set forth in SEQ ID NOs: 19 and 33; 20and 34; 21 and 35; 23 and 37; 24 and 37; 25 and 37; 26 and 37; 27 and37; 28 and 37; 29 and 37; 30 and 37; 23 and 38; 24 and 38; 25 and 38; 26and 38; 27 and 38; 28 and 38; 23 and 39; or, 23 and 40, respectively,and wherein each CDR is defined in accordance with the MacCallumdefinition, the Kabat definition, the Chothia definition, the IMGTnumbering system, the AbM definition of CDR, structural analysis, or acombination thereof, wherein the structural analysis identifies residuesin the variable region(s) predicted to make contact with an epitoperegion of CD73 (e.g., human, mouse, or cynomolgus CD73). In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73) andcomprises a combination of CDRs defined by the Kabat definition and CDRsdefined by structural analysis of the antibody, wherein the structuralanalysis identifies residues in the variable region(s) predicted to makecontact with an epitope region of CD73 (e.g., human, mouse, orcynomolgus CD73).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising the six CDRs present in theBA025 antibody disclosed herein, as defined by one or more of the Kabat,IMGT (e.g., IMGT unique), AHo or Chothia antibody numbering schemes. Incertain embodiments, the antibody comprises at least one, at least two,at least three, at least four, or at least five of the CDRs present inthe BA025 antibody. The Kabat, IMGT unique, Aho, and Chothia CDRs of theBA025 antibody are provided in Table 1 herein.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising the six CDRs present in theBA020 antibody disclosed herein, as defined by one or more of the Kabat,IMGT (e.g., IMGT unique), AHo or Chothia antibody numbering schemes. Incertain embodiments, the antibody comprises at least one, at least two,at least three, at least four, or at least five of the CDRs present inthe BA020 antibody. The Kabat, IMGT unique, Aho, and Chothia CDRs of theBA020 antibody are provided in Table 1 herein.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising:

-   -   (a) CDRH1 comprises the amino acid sequence of X₁X₂WX₃X₄(SEQ ID        NO: 13), wherein        -   X₁ is S or N;        -   X₂ is S or Y;        -   X₃ is I or M; and        -   X₄ is N or H;    -   (b) CDRH2 comprises the amino acid sequence of        X₁IYPRX₂X₃DTNYX₄X₅KFKX₆ (SEQ ID NO: 14), wherein        -   X₁ is R or T;        -   X₂ is N, A, or S;        -   X₃ is G or S;        -   X₄ is N, A, or S;        -   X₅ is G or Q; and        -   X₆ is D or G;    -   (c) CDRH3 comprises the amino acid sequence of LLDYSMDY (SEQ ID        NO: 7);    -   (d) CDRL1 comprises the amino acid sequence of RASQDISX₁X₂LN        (SEQ ID NO: 16), wherein        -   X₁ is N or I; and        -   X₂ is Y or S;    -   (e) CDRL2 comprises the amino acid sequence of YTSRLHS (SEQ ID        NO: 10); and/or    -   (f) CDRL3 comprises the amino acid sequence of QQGNTLPXT (SEQ ID        NO: 17), wherein        -   X is L or W.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising:

-   -   (a) CDRH1 comprises the amino acid sequence of SSWIN (SEQ ID NO:        2);    -   (b) CDRH2 comprises the amino acid sequence of        RIYPRX₁GDTNYX₂GKFKD (SEQ ID NO: 15), wherein        -   X₁ is N, A, or S; and        -   X₂ is N, A, or S;    -   (c) CDRH3 comprises the amino acid sequence of LLDYSMDY (SEQ ID        NO: 7);    -   (d) CDRL1 comprises the amino acid sequence of RASQDISX1X2LN        (SEQ ID NO: 16), wherein        -   X₁ is N or I; and        -   X₂ is Y or S;    -   (e) CDRL2 comprises the amino acid sequence of YTSRLHS (SEQ ID        NO: 10); and/or    -   (f) CDRL3 comprises the amino acid sequence of QQGNTLPLT (SEQ ID        NO: 12)

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), wherein the antibody comprises a VH domain comprisingthe CDRH1, CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID NOs:1, 3, and 7, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), wherein the antibody comprisesa VH domain comprising the CDRH1, CDRH2 and CDRH3 amino acid sequencesset forth in SEQ ID NOs: 2, 4, and 7, respectively. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73),wherein the antibody comprises a VH domain comprising the CDRH1, CDRH2and CDRH3 amino acid sequences set forth in SEQ ID NOs: 2, 5, and 7,respectively. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), wherein the antibody comprises a VH domaincomprising the CDRH1, CDRH2 and CDRH3 amino acid sequences set forth inSEQ ID NOs: 2, 6, and 7, respectively. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), wherein the antibodycomprises a VL domain comprising the CDRL1, CDRL2 and CDRL3 amino acidsequences set forth in SEQ ID NOs: 8, 10, and 11, respectively. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), wherein the antibody comprises a VL domain comprisingthe CDRL1, CDRL2 and CDRL3 amino acid sequences set forth in SEQ ID NOs:9, 10, and 12, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), wherein the antibody comprisesa VL domain comprising the CDRL1, CDRL2 and CDRL3 amino acid sequencesset forth in SEQ ID NOs: 8, 10, and 12, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), wherein the antibody comprises a heavy chain variableregion comprising CDRH1, CDRH2, and CDRH3 regions, and a light chainvariable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein theCDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the aminoacid sequences set forth in SEQ ID NOs: 1, 3, 7, 8, 10, and 11,respectively. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), wherein the antibody comprises a heavy chainvariable region comprising CDRH1, CDRH2, and CDRH3 regions, and a lightchain variable region comprising CDRL1, CDRL2, and CDRL3 regions,wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regionscomprise the amino acid sequences set forth in SEQ ID NOs: 2, 4, 7, 8,10, and 11, respectively. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD73 (e.g.,human, mouse, or cynomolgus CD73), wherein the antibody comprises aheavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions,and a light chain variable region comprising CDRL1, CDRL2, and CDRL3regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3regions comprise the amino acid sequences set forth in SEQ ID NOs: 2, 4,7, 8, 10, and 12, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), wherein the antibody comprisesa heavy chain variable region comprising CDRH1, CDRH2, and CDRH3regions, and a light chain variable region comprising CDRL1, CDRL2, andCDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3regions comprise the amino acid sequences set forth in SEQ ID NOs: 2, 4,7, 9, 10, and 12, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), wherein the antibody comprisesa heavy chain variable region comprising CDRH1, CDRH2, and CDRH3regions, and a light chain variable region comprising CDRL1, CDRL2, andCDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3regions comprise the amino acid sequences set forth in SEQ ID NOs: 2, 5,7, 8, 10, and 12, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), wherein the antibody comprisesa heavy chain variable region comprising CDRH1, CDRH2, and CDRH3regions, and a light chain variable region comprising CDRL1, CDRL2, andCDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3regions comprise the amino acid sequences set forth in SEQ ID NOs: 2, 6,7, 8, 10, and 12, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), wherein the antibody comprisesa heavy chain variable region comprising CDRH1, CDRH2, and CDRH3regions, and a light chain variable region comprising CDRL1, CDRL2, andCDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3regions comprise the amino acid sequences set forth in SEQ ID NOs: 2, 5,7, 9, 10, and 12, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), wherein the antibody comprisesa heavy chain variable region comprising CDRH1, CDRH2, and CDRH3regions, and a light chain variable region comprising CDRL1, CDRL2, andCDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3regions comprise the amino acid sequences set forth in SEQ ID NOs: 2, 6,7, 9, 10, and 12, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 31. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), comprising a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 19-21 and 23-30. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73),comprising a heavy chain variable region comprising an amino acidsequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., atleast 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 19-21 and 23-30.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 41 or 42. In certain embodiments,the instant disclosure provides an isolated antibody that specificallybinds to CD73 (e.g., human, mouse, or cynomolgus CD73), comprising alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 33-35 and 37-40. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73),comprising a light chain variable region comprising an amino acidsequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., atleast 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 33-35 and 37-40.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 31 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 41 or 42. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:19-21 and 23-30, and a light chain variable region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 33-35and 37-40. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), comprising a heavy chain variable region comprisingan amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100%(e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or99%) identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19-21 and 23-30, and a light chain variableregion comprising an amino acid sequence that is at least 75%, 80%, 85%,90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99%) identical to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 33-35 and 37-40.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region and a lightchain variable region comprising the amino acid sequences set forth inSEQ ID NOs: 19 and 33; 20 and 34; 21 and 35; 23 and 37; 24 and 37; 25and 37; 26 and 37; 27 and 37; 28 and 37; 29 and 37; 30 and 37; 23 and38; 24 and 38; 25 and 38; 26 and 38; 27 and 38; 28 and 38; 23 and 39;or, 23 and 40, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), comprising a heavy chainvariable region comprising an amino acid sequence of SEQ ID NO: 19, anda light chain variable region comprising an amino acid sequence of SEQID NO: 33. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), comprising a heavy chain variable region comprisingan amino acid sequence of SEQ ID NO: 20, and a light chain variableregion comprising an amino acid sequence of SEQ ID NO: 34. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 21, and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 35. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), comprising a heavychain variable region comprising an amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising an amino acid sequenceof SEQ ID NO: 37. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD73 (e.g.,human, mouse, or cynomolgus CD73), comprising a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO: 24, and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:37. In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprising anamino acid sequence of SEQ ID NO: 23, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO: 38. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 24, and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 38. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), comprising a heavychain variable region comprising an amino acid sequence of SEQ ID NO:25, and a light chain variable region comprising an amino acid sequenceof SEQ ID NO: 37. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD73 (e.g.,human, mouse, or cynomolgus CD73), comprising a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO: 26, and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:37. In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprising anamino acid sequence of SEQ ID NO: 27, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO: 37. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 28, and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 37. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), comprising a heavychain variable region comprising an amino acid sequence of SEQ ID NO:25, and a light chain variable region comprising an amino acid sequenceof SEQ ID NO: 38. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD73 (e.g.,human, mouse, or cynomolgus CD73), comprising a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO: 26, and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:38. In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprising anamino acid sequence of SEQ ID NO: 27, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO: 38. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 28, and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 38. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), comprising a heavychain variable region comprising an amino acid sequence of SEQ ID NO:29, and a light chain variable region comprising an amino acid sequenceof SEQ ID NO: 37. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD73 (e.g.,human, mouse, or cynomolgus CD73), comprising a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO: 30, and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:37. In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprising anamino acid sequence of SEQ ID NO: 23, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO: 39. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CD73 (e.g., human, mouse, or cynomolgus CD73),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 23, and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 40.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprising anamino acid sequence derived from a murine IgHV1S12 germline sequence. Incertain embodiments, the murine IgHV1S12 germline sequence is set forthin SEQ ID NO: 18. One or more regions selected from framework 1,framework 2, framework 3, CDRH1, and CDRH2 (e.g., two, three, four orfive of these regions) can be derived from a murine IgHV1S12 germlinesequence. In one embodiment, framework 1, framework 2, framework 3,CDRH1, and CDRH2 are all derived from a murine IgHV1S12 germlinesequence. In certain embodiments, the heavy chain variable regioncomprises a CDRH3 comprising the amino acid sequence set forth in SEQ IDNO: 7.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a light chain variable region comprising anamino acid sequence derived from a murine IgKV10-96 germline sequence.In certain embodiments, the murine IgKV10-96 germline sequence is setforth in SEQ ID NO: 32. One or more regions selected from framework 1,framework 2, framework 3, CDRL1, and CDRL2 (e.g., two, three, four orfive of these regions) can be derived from a murine germline sequence ofIgKV10-96. In one embodiment, framework 1, framework 2, framework 3,CDRL1, and CDRL2 are all derived from a murine germline sequence ofIgKV10-96. In certain embodiments, the light chain variable regioncomprises a CDRL3 comprising the amino acid sequence set forth in SEQ IDNO: 11 or 12.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprising anamino acid sequence derived from a murine IgHV1S12 germline sequence(e.g., SEQ ID NO: 18); and a light chain variable region comprising anamino acid sequence derived from a murine germline sequence derived froma murine IgKV10-96 germline sequence (e.g., SEQ ID NO: 32). In certainembodiments, the heavy chain variable region comprises a CDRH3comprising the amino acid sequence set forth in SEQ ID NO: 7, and thelight chain variable region comprises a CDRL3 comprising the amino acidsequence set forth in SEQ ID NO: 11 or 12.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprising anamino acid sequence derived from a human IgHV5-51 germline sequence. Incertain embodiments, the human IgHV5-51germline sequence is a humanIgHV5-51*02 germline sequence (e.g., comprising the amino acid sequenceof SEQ ID NO: 22). One or more regions selected from framework 1,framework 2, framework 3, CDRH1, and CDRH2 (e.g., two, three, four orfive of these regions) can be derived from a human IgHV5-51 germlinesequence. In one embodiment, framework 1, framework 2, framework 3,CDRH1, and CDRH2 are all derived from a human IgHV5-51 germlinesequence. In certain embodiments, the heavy chain variable regioncomprises a CDRH3 comprising the amino acid sequence set forth in SEQ IDNO: 7.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a light chain variable region comprising anamino acid sequence derived from a human IGKV1-33 germline sequence(e.g., IGKV1-33*01, e.g., comprising the amino acid sequence of SEQ IDNO: 36). One or more regions selected from framework 1, framework 2,framework 3, CDRL1, and CDRL2 (e.g., two, three, four or five of theseregions) can be derived from a human germline sequence selected from thegroup consisting of IGKV1-33 (e.g., IGKV1-33*01, e.g., comprising theamino acid sequence of SEQ ID NO: 36). In one embodiment, framework 1,framework 2, framework 3, CDRL1, and CDRL2 are all derived from a humanIGKV1-33 germline sequence (e.g., IGKV1-33*01, e.g., comprising theamino acid sequence of SEQ ID NO: 36). In certain embodiments, the lightchain variable region comprises a CDRL3 comprising the amino acidsequence set forth in SEQ ID NO: 12.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), comprising a heavy chain variable region comprising anamino acid sequence derived from a human IgHV5-51 germline sequence(e.g., a human IgHV5-51*02 germline sequence, e.g., comprising the aminoacid sequence of SEQ ID NO: 22); and a light chain variable regioncomprising an amino acid sequence derived from a human IGKV1-33 germlinesequence (e.g., IGKV1-33*01, e.g., comprising the amino acid sequence ofSEQ ID NO: 36). In certain embodiments, the heavy chain variable regioncomprises a CDRH3 comprising the amino acid sequence set forth in SEQ IDNO: 7, and the light chain variable region comprises a CDRL3 comprisingthe amino acid sequence set forth in SEQ ID NO: 12.

In certain embodiments, the instant disclosure provides an isolatedantibody that cross-competes for binding to CD73 (e.g., human, mouse, orcynomolgus CD73) with any of the anti-CD73 antibodies disclosed herein,including an antibody comprising the heavy chain variable region andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 19 and 33; 20 and 34; 21 and 35; 23 and 37; 24 and 37; 25 and 37;26 and 37; 27 and 37; 28 and 37; 29 and 37; 30 and 37; 23 and 38; 24 and38; 25 and 38; 26 and 38; 27 and 38; 28 and 38; 23 and 39; or, 23 and40, respectively. In certain embodiments, the instant disclosureprovides an isolated antibody that cross-competes for binding to CD73(e.g., human, mouse, or cynomolgus CD73) with an antibody comprising theheavy and light chain variable region amino acid sequences set forth inSEQ ID NOs: 24 and 37, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that cross-competes for bindingto CD73 (e.g., human, mouse, or cynomolgus CD73) with an antibodycomprising the heavy and light chain variable region amino acidsequences set forth in SEQ ID NOs: 27 and 37, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that binds to the same or an overlapping epitope of CD73 (e.g.,an epitope of human, mouse, or cynomolgus CD73) as an antibody describedherein, e.g., an antibody comprising the heavy and light chain variableregion amino acid sequences set forth in SEQ ID NOs: 19 and 33; 20 and34; 21 and 35; 23 and 37; 24 and 37; 25 and 37; 26 and 37; 27 and 37; 28and 37; 29 and 37; 30 and 37; 23 and 38; 24 and 38; 25 and 38; 26 and38; 27 and 38; 28 and 38; 23 and 39; or, 23 and 40, respectively. Incertain embodiments, the instant disclosure provides an isolatedantibody that binds to the same or an overlapping epitope of CD73 (e.g.,an epitope of human, mouse, or an epitope of cynomolgus CD73) as anantibody described herein, e.g., an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 24 and 37, respectively. In certain embodiments, the instantdisclosure provides an isolated antibody that binds to the same or anoverlapping epitope of CD73 (e.g., an epitope of human, mouse, or anepitope of cynomolgus CD73) as an antibody described herein, e.g., anantibody comprising the heavy and light chain variable region amino acidsequences set forth in SEQ ID NOs: 27 and 37, respectively. In certainembodiments, the epitope of an antibody can be determined by, e.g., NMRspectroscopy, surface plasmon resonance (BIAcore®), X-ray diffractioncrystallography studies, ELISA assays, hydrogen/deuterium exchangecoupled with mass spectrometry (e.g., liquid chromatography electrospraymass spectrometry), array-based oligo-peptide scanning assays, and/ormutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-raycrystallography, crystallization may be accomplished using any of theknown methods in the art (e.g., Giegé R et al., (1994) Acta CrystallogrD Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem189: 1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976)J Biol Chem 251: 6300-6303, all of which are herein incorporated byreference in their entireties). Antibody:antigen crystals may be studiedusing well known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114& 115, eds Wyckoff H W et al.; U.S. Patent Application No.2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D BiolCrystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323, all of which are herein incorporatedby reference in their entireties). Mutagenesis mapping studies may beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) supra and Cunningham B C & Wells J A(1989) supra for a description of mutagenesis techniques, includingalanine scanning mutagenesis techniques. In a specific embodiment, theepitope of an antibody is determined using alanine scanning mutagenesisstudies. In addition, antibodies that recognize and bind to the same oroverlapping epitopes of CD73 (e.g., human, mouse, or cynomolgus CD73)can be identified using routine techniques such as an immunoassay, forexample, by showing the ability of one antibody to block the binding ofanother antibody to a target antigen, i.e., a competitive binding assay.Competition binding assays also can be used to determine whether twoantibodies have similar binding specificity for an epitope. Competitivebinding can be determined in an assay in which the immunoglobulin undertest inhibits specific binding of a reference antibody to a commonantigen, such as CD73 (e.g., human, mouse, or cynomolgus CD73). Numeroustypes of competitive binding assays are known, for example: solid phasedirect or indirect radioimmunoassay (RIA), solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay (seeStahli C et al., (1983) Methods Enzymol 9: 242-253); solid phase directbiotin-avidin EIA (see Kirkland T N et al., (1986) J Immunol 137:3614-9); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow E & Lane D, (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Press); solid phase direct label RIA usingI-125 label (see Morel G A et al., (1988) Mol Immunol 25(1): 7-15);solid phase direct biotin-avidin EIA (see Cheung R C et al., (1990)Virology 176: 546-52); and direct labeled RIA (see Moldenhauer G et al.,(1990) Scand J Immunol 32: 77-82), all of which are herein incorporatedby reference in their entireties. Typically, such an assay involves theuse of purified antigen (e.g., CD73, such as human, mouse, or cynomolgusCD73) bound to a solid surface or cells bearing either of these, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition can be measured by determining the amount oflabel bound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. A competition bindingassay can be configured in a large number of different formats usingeither labeled antigen or labeled antibody. In a common version of thisassay, the antigen is immobilized on a 96-well plate. The ability ofunlabeled antibodies to block the binding of labeled antibodies to theantigen is then measured using radioactive or enzyme labels. For furtherdetails see, for example, Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274;Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al.,(1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane Deditors supra, pp. 386-389, all of which are herein incorporated byreference in their entireties.

Any immunoglobulin (Ig) constant region can be used in the antibodiesdisclosed herein. In certain embodiments, the Ig region is a human IgG,IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g.,IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂), or any subclass (e.g., IgG_(2a)and IgG_(2b)) of immunoglobulin molecule.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising a heavy chain constant regioncomprising the amino acid sequence of SEQ ID NO: 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, or 58. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a light chain constant region comprising the amino acidsequence of SEQ ID NO: 89 or 93.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system, to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425, herein incorporated by reference in itsentirety. The number of cysteine residues in the hinge region of the CH1domain may be altered to, e.g., facilitate assembly of the light andheavy chains, or to alter (e.g., increase or decrease) the stability ofthe antibody.

In a specific embodiment, one, two, or more amino acid mutations (e.g.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fe orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745, all of which are hereinincorporated by reference in their entireties, for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo. In certain embodiments, one, two or more amino acidmutations (e.g., substitutions, insertions, or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fe or hinge-Fc domain fragment) to decrease the half-lifeof the antibody in vivo. In other embodiments, one, two or more aminoacid mutations (e.g., substitutions, insertions or deletions) areintroduced into an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fe or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In a specific embodiment, the antibodies mayhave one or more amino acid mutations (e.g., substitutions) in thesecond constant (CH2) domain (residues 231-340 of human IgG₁) and/or thethird constant (CH3) domain (residues 341-447 of human IgG₁), numberedaccording to the EU numbering system. In a specific embodiment, theconstant region of the IgG₁ of an antibody described herein comprises amethionine (M) to tyrosine (Y) substitution in position 252, a serine(S) to threonine (T) substitution in position 254, and a threonine (T)to glutamic acid (E) substitution in position 256, numbered according tothe EU numbering system. See U.S. Pat. No. 7,658,921, which is hereinincorporated by reference in its entirety. This type of mutant IgG,referred to as “YTE mutant” has been shown to display four foldincreased half-life as compared to wild-type versions of the sameantibody (see Dall'Acqua W F et al., (2006) J Biol Chem 281: 23514-24,which is herein incorporated by reference in its entirety). In certainembodiments, an antibody comprises an IgG constant domain comprisingone, two, three or more amino acid substitutions of amino acid residuesat positions 251-257, 285-290, 308-314, 385-389, and 428-436, numberedaccording to the EU numbering system.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system, to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, all of which are hereinincorporated by reference in their entireties.

In certain embodiments, the antibody comprises a heavy chain constantregion that is a variant of a wild-type heavy chain constant region,wherein the variant heavy chain constant region binds to FcγRIIB withhigher affinity than the wild-type heavy chain constant region binds toFcγRIIB. In certain embodiments, the variant heavy chain constant regionis a variant human heavy chain constant region, e.g., a variant humanIgG₁, a variant human IgG₂, or a variant human IgG₄ heavy chain constantregion. In certain embodiments, the variant human IgG heavy chainconstant region comprises one or more of the following amino acidmutations, according to the EU numbering system: G236D, P238D, S239D,S267E, L328F, and L328E. In certain embodiments, the variant human IgGheavy chain constant region comprises a set of amino acid mutationsselected from the group consisting of: S267E and L328F; P238D and L328E;P238D and one or more substitutions selected from the group consistingof E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D,P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, andL328F; and V264E, S267E, and L328F, according to the EU numberingsystem. In certain embodiments, the FcγRIIB is expressed on a cellselected from the group consisting of macrophages, monocytes, B cells,dendritic cells, endothelial cells, and activated T cells.

In a further embodiment, one, two, or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 239, 243, 267,292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered accordingto the EU numbering system, can be replaced with a different amino acidresidue such that the antibody has an altered affinity for an effectorligand but retains the antigen-binding ability of the parent antibody.The effector ligand to which affinity is altered can be, for example, anFc receptor or the C1 component of complement. This approach isdescribed in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260,each of which is herein incorporated by reference in its entirety. Incertain embodiments, the deletion or inactivation (through pointmutations or other means) of a constant region domain may reduce Fcreceptor binding of the circulating antibody thereby increasing tumorlocalization. See, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886, each ofwhich is herein incorporated by reference in its entirety, for adescription of mutations that delete or inactivate the constant domainand thereby increase tumor localization. In certain embodiments, one ormore amino acid substitutions may be introduced into the Fc region of anantibody described herein to remove potential glycosylation sites on theFc region, which may reduce Fc receptor binding (see, e.g., Shields R Let al., (2001) J Biol Chem 276: 6591-604, which is herein incorporatedby reference in its entirety). In various embodiments, one or more ofthe following mutations in the constant region of an antibody describedherein may be made: an N297A substitution; an N297Q substitution; anL234A substitution; an L234F substitution; an L235A substitution; anL235F substitution; an L235V substitution; an L237A substitution; anS239D substitution; an E233P substitution; an L234V substitution; anL235A substitution; a C236 deletion; a P238A substitution; an S239Dsubstitution; an F243L substitution; a D265A substitution; an S267Esubstitution; an L328F substitution; an R292P substitution; a Y300Lsubstitution; an A327Q substitution; a P329A substitution; an A332Lsubstitution; an 1332E substitution; or a P396L substitution, numberedaccording to the EU numbering system.

In certain embodiments, a mutation selected from the group consisting ofD265A, P329A, and a combination thereof, numbered according to the EUnumbering system, may be made in the constant region of an antibodydescribed herein. In certain embodiments, a mutation selected from thegroup consisting of L235A, L237A, and a combination thereof, numberedaccording to the EU numbering system, may be made in the constant regionof an antibody described herein. In certain embodiments, a mutationselected from the group consisting of S267E, L328F, and a combinationthereof, numbered according to the EU numbering system, may be made inthe constant region of an antibody described herein. In certainembodiments, a mutation selected from the group consisting of S239D,1332E, optionally A330L, and a combination thereof, numbered accordingto the EU numbering system, may be made in the constant region of anantibody described herein. In certain embodiments, a mutation selectedfrom the group consisting of L235V, F243L, R292P, Y300L, P396L, and acombination thereof, numbered according to the EU numbering system, maybe made in the constant region of an antibody described herein. Incertain embodiments, a mutation selected from the group consisting ofS267E, L328F, and a combination thereof, numbered according to the EUnumbering system, may be made in the constant region of an antibodydescribed herein.

In a specific embodiment, an antibody described herein comprises theconstant domain of an IgG₁ with an N297Q or N297A amino acidsubstitution, numbered according to the EU numbering system. In oneembodiment, an antibody described herein comprises the constant domainof an IgG₁ with a mutation selected from the group consisting of D265A,P329A, and a combination thereof, numbered according to the EU numberingsystem. In another embodiment, an antibody described herein comprisesthe constant domain of an IgG₁ with a mutation selected from the groupconsisting of L234A, L235A, and a combination thereof, numberedaccording to the EU numbering system. In another embodiment, an antibodydescribed herein comprises the constant domain of an IgG₁ with amutation selected from the group consisting of L234F, L235F, N297A, anda combination thereof, numbered according to the EU numbering system. Incertain embodiments, amino acid residues in the constant region of anantibody described herein in the positions corresponding to positionsL234, L235, and D265 in a human IgG₁ heavy chain, numbered according tothe EU numbering system, are not L, L, and D, respectively. Thisapproach is described in detail in International Publication No. WO14/108483, which is herein incorporated by reference in its entirety. Ina particular embodiment, the amino acids corresponding to positionsL234, L235, and D265 in a human IgG₁ heavy chain are F, E, and A; or A,A, and A, respectively, numbered according to the EU numbering system.

In certain embodiments, one or more amino acids selected from amino acidresidues 329, 331, and 322 in the constant region of an antibodydescribed herein, numbered according to the EU numbering system, can bereplaced with a different amino acid residue such that the antibody hasaltered C1q binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al.), which is herein incorporated byreference in its entirety. In certain embodiments, one or more aminoacid residues within amino acid positions 231 to 238 in the N-terminalregion of the CH2 domain of an antibody described herein are altered tothereby alter the ability of the antibody to fix complement, numberedaccording to the EU numbering system. This approach is described furtherin International Publication No. WO 94/29351, which is hereinincorporated by reference in its entirety. In certain embodiments, theFc region of an antibody described herein is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fey receptor by mutating one or more amino acids (e.g., introducingamino acid substitutions) at the following positions: 238, 239, 248,249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278,280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303,305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331,333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398,414, 416, 419, 430, 434, 435, 437, 438, or 439, numbered according tothe EU numbering system. This approach is described further inInternational Publication No. WO 00/42072, which is herein incorporatedby reference in its entirety.

In certain embodiments, an antibody described herein comprises amodified constant domain of an IgG₁, wherein the modification increasesthe ability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC). In certain embodiments, 0.1, 1, or 10 μg/ml of theantibody is capable of inducing cell death of at least 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, or 60% of CD73-expressing cells within 1, 2, or3 hours, as assessed by methods described herein and/or known to aperson of skill in the art. In certain embodiments, the modifiedconstant domain of an IgG₁ comprises S239D and 1332E substitutions,numbered according to the EU numbering system. In certain embodiments,the modified constant domain of an IgG₁ comprises S239D, A330L, and1332E substitutions, numbered according to the EU numbering system. Incertain embodiments, the modified constant domain of an IgG₁ comprisesL235V, F243L, R292P, Y300L, and P396L substitutions, numbered accordingto the EU numbering system. In certain embodiments, the antibody iscapable of inducing cell death in effector T cells and Tregs, whereinthe percentage of Tregs that undergo cell death is higher than thepercentage of effector T cells that undergo cell death by at least 1.2fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, or 5 fold.

In certain embodiments, an antibody described herein comprises theconstant region of an IgG₄ antibody and the serine at amino acid residue228 of the heavy chain, numbered according to the EU numbering system,is substituted for proline. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73), the antibody comprising aheavy chain constant region comprising the amino acid sequence of SEQ IDNO: 26.

In certain embodiments, any of the constant region mutations ormodifications described herein can be introduced into one or both heavychain constant regions of an antibody described herein having two heavychain constant regions.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising a heavy chain comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:63-88. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO: 63. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 64. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 65. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 66. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 67. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 68. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 69. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 70. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 71. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 72. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 73. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 74. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 75. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 76. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 77. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 78. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 79. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 80. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 81. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 82. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 83. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 84. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 85. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 86. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 87. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 88.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising a light chain comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:92-98. In certain embodiments, the antibody comprises a light chaincomprising the amino acid sequence set forth in SEQ ID NO: 92. Incertain embodiments, the antibody comprises a light chain comprising theamino acid sequence set forth in SEQ ID NO: 93. In certain embodiments,the antibody comprises a light chain comprising the amino acid sequenceset forth in SEQ ID NO: 94. In certain embodiments, the antibodycomprises a light chain comprising the amino acid sequence set forth inSEQ ID NO: 95. In certain embodiments, the antibody comprises a lightchain comprising the amino acid sequence set forth in SEQ ID NO: 96. Incertain embodiments, the antibody comprises a light chain comprising theamino acid sequence set forth in SEQ ID NO: 97. In certain embodiments,the antibody comprises a light chain comprising the amino acid sequenceset forth in SEQ ID NO: 98.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73), the antibody comprising a heavy chain comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:63-88; and a light chain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 92-98. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 63; and a light chain comprising the amino acid sequence of SEQ IDNO: 92. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 64; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 92. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 65; and a light chain comprising the amino acid sequence of SEQ IDNO: 94. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 66; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 94. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 67; and a light chain comprising the amino acid sequence of SEQ IDNO: 95. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 68; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 95. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 69; and a light chain comprising the amino acid sequence of SEQ IDNO: 96. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 70; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 96. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 71; and a light chain comprising the amino acid sequence of SEQ IDNO: 96. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 72; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 96. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 73; and a light chain comprising the amino acid sequence of SEQ IDNO: 96. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 74; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 96. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 75; and a light chain comprising the amino acid sequence of SEQ IDNO: 96. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 76; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 96. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 77; and a light chain comprising the amino acid sequence of SEQ IDNO: 97. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 78; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 97. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 79; and a light chain comprising the amino acid sequence of SEQ IDNO: 97. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 80; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 97. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 77; and a light chain comprising the amino acid sequence of SEQ IDNO: 98. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 78; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 98. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 79; and a light chain comprising the amino acid sequence of SEQ IDNO: 98. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 80; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 98. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 81; and a light chain comprising the amino acid sequence of SEQ IDNO: 97. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 82; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 97. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 83; and a light chain comprising the amino acid sequence of SEQ IDNO: 97. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 84; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 97. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 85; and a light chain comprising the amino acid sequence of SEQ IDNO: 97. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 86; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 97. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 87; and a light chain comprising the amino acid sequence of SEQ IDNO: 97. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 88; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 97. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 81; and a light chain comprising the amino acid sequence of SEQ IDNO: 98. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 82; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 98. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 83; and a light chain comprising the amino acid sequence of SEQ IDNO: 98. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 84; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 98. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 85; and a light chain comprising the amino acid sequence of SEQ IDNO: 98. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 86; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 98. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 87; and a light chain comprising the amino acid sequence of SEQ IDNO: 98. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73), the antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 88; and a light chain comprisingthe amino acid sequence of SEQ ID NO: 98.

Any antibody format can be used in the antibodies disclosed herein. Incertain embodiments, the antibody is a single chain antibody orsingle-chain Fv (scFv). In certain embodiments, the antibody is a scFvfused with an Fc region (scFv-Fc). In certain embodiments, the antibodyis a Fab fragment. In certain embodiments, the antibody is a F(ab′)₂fragment.

In certain embodiments, the antibody disclosed herein is a multispecificantibody (e.g., a bispecific antibody) which specifically binds to CD73(e.g., human, mouse, or cynomolgus CD73) and a second antigen.

In certain embodiments, the antibody disclosed herein is conjugated to asecond antibody that specifically binds to a second antigen. In certainembodiments, the antibody disclosed herein is covalently conjugated to asecond antibody. In certain embodiments, the antibody disclosed hereinis non-covalently conjugated to a second antibody. In certainembodiments, the antibody disclosed herein is cross-linked to a secondantibody. In certain embodiments, the second antigen is atumor-associated antigen (e.g., a polypeptide overexpressed in a tumor,a polypeptide derived from an oncovirus, a polypeptide comprising apost-translational modification specific to a tumor, a polypeptidespecifically mutated in a tumor). In certain embodiments, thetumor-associated antigen is EGFR (e.g., human EGFR), optionally whereinthe second antibody is cetuximab. In certain embodiments, thetumor-associated antigen is Her2 (e.g., human Her2), optionally whereinthe second antibody is trastuzumab. In certain embodiments, thetumor-associated antigen is CD20 (e.g., human CD20).

In certain embodiments, the antibody disclosed herein is conjugated to acytotoxic agent, cytostatic agent, toxin, radionuclide, or detectablelabel. In certain embodiments, the cytotoxic agent is able to inducedeath or destruction of a cell in contact therewith. In certainembodiments, the cytostatic agent is able to prevent or substantiallyreduce proliferation and/or inhibits the activity or function of a cellin contact therewith. In certain embodiments, the cytotoxic agent orcytostatic agent is a chemotherapeutic agent. In certain embodiments,the radionuclide is selected from the group consisting of the isotopes³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹¹⁷Lu, ¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Ac and ¹⁸⁶Re. Incertain embodiments, the detectable label comprises a fluorescent moietyor a click chemistry handle.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73) and functions as an antagonist (e.g., decreases orinhibits CD73 activity).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73) and decreases or inhibits CD73 (e.g., human, mouse, orcynomolgus CD73) activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%,as assessed by methods described herein and/or known to one of skill inthe art, relative to CD73 (e.g., human, mouse, or cynomolgus CD73)activity without any antibody or with an unrelated antibody (e.g., anantibody that does not specifically bind to CD73 (e.g., human, mouse, orcynomolgus CD73)). In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD73 (e.g.,human, mouse, or cynomolgus CD73) and decreases or inhibits CD73 (e.g.,human, mouse, or cynomolgus CD73) activity by at least about 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, 100 fold, or more, as assessed by methods described herein and/orknown to one of skill in the art, relative to CD73 (e.g., human, mouse,or cynomolgus CD73) activity without any antibody or with an unrelatedantibody (e.g., an antibody that does not specifically bind to CD73(e.g., human CD73)). Non-limiting examples of CD73 (e.g., human, mouse,or cynomolgus CD73) activity can include CD73 (e.g., human, mouse, orcynomolgus CD73) signaling; CD73 (e.g., human, mouse, or cynomolgusCD73) binding to its binding partners and/or substrates (e.g., AMP or ananalog or variant thereof); CD73 catalyzing the conversion of at leastone of its substrates (e.g., AMP or an analog or variant thereof),facilitation of tumor growth and metastasis, and other known functions(e.g., see Gao et al. (2014) BioMed Res. Int. Article ID 460654). Inspecific embodiments, reduction in a CD73 (e.g., human, mouse, orcynomolgus CD73) activity is assessed as described in the Examples,infra.

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73) and decreases or inhibits CD73 (e.g., human, mouse, orcynomolgus CD73) enzymatic activity (e.g., catalyzing the conversion ofAMP into adenosine) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%,as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, relative to CD73 (e.g., human, mouse,or cynomolgus CD73) enzymatic activity without any antibody or with anunrelated antibody (e.g., an antibody that does not specifically bind toCD73 (e.g., human, mouse, or cynomolgus CD73)). In specific embodiments,the instant disclosure provides an isolated antibody that specificallybinds to CD73 (e.g., human, mouse, cynomolgus CD73) and decreases orinhibits CD73 (e.g., human, mouse, cynomolgus CD73) enzymatic activity(e.g., catalyzing the conversion of AMP into adenosine) by at leastabout 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold,3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, or 100 fold, as assessed by methods described herein (seethe Examples, infra) or known to one of skill in the art, relative toCD73 (e.g., human CD73) enzymatic activity without any antibody or withan unrelated antibody (e.g., an antibody that does not specifically bindto CD73 (e.g., human, mouse, or cynomolgus CD73)).

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CD73 (e.g., human, mouse, orcynomolgus CD73) and reduces cancer progression (e.g., proliferationand/or metastasis of cells in the microenvironment of a cancer typedisclosed herein) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%,as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, relative to the cancer progressionwithout any antibody or with an unrelated antibody (e.g., an antibodythat does not specifically bind to CD73 (e.g., human, mouse, orcynomolgus CD73)). In specific embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CD73 (e.g.,human, mouse, or cynomolgus CD73) and reduces cancer progression (e.g.,proliferation and/or metastasis of cells in the microenvironment of acancer type disclosed herein) by at least about 1.2 fold, 1.3 fold, 1.4fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold,or more, as assessed by methods described herein (see the Examples,infra) or known to one of skill in the art, relative to the cancerprogression without any antibody or with an unrelated antibody (e.g., anantibody that does not specifically bind to CD73 (e.g., human, mouse, orcynomolgus CD73)). 5.3 Anti-CD73 antibodies comprising ligand bindingmoieties

In one aspect, the instant disclosure provides antibodies thatspecifically bind to CD73 (e.g., human CD73, cynomolgus CD73, or mouseCD73) and comprise a ligand binding moiety (e.g., a TGFβ-binding moietyor a VEGF-binding moiety).

Anti-CD73 antibodies comprising a ligand binding moiety can comprise animmunoglobulin chain of any anti-CD73 antibody. Exemplary anti-CD73antibodies are disclosed in Section 5.2 of the instant specification.Additional exemplary anti-CD73 antibodies are disclosed inWO2016055609A1, WO2016075099A1, WO2016081748A2, WO2016131950A1,WO2017064043A1, WO2017100670A1, WO2017118613A1, WO2017152085A1, and U.S.Pat. No. 9,388,249B2, each of which is incorporated herein by referencein its entirety. In certain embodiments, the anti-CD73 antibodycomprises a VH and a VL of an anti-CD73 antibody as disclosed in Section5.2. In certain embodiments, the anti-CD73 antibody comprises a heavychain and a light chain of an anti-CD73 antibody as disclosed in Section5.2.

A ligand binding moiety can be linked to any portion of animmunoglobulin chain of an anti-CD73 antibody, including the N- orC-terminal amino acid residue. The ligand binding moiety can be linked(covalently or non-covalently) to the immunoglobulin chain directly orvia a linker (e.g., a peptide linker). Covalent linkage can be achemical linkage or a genetic linkage (i.e., to form a fusion protein).In certain embodiments, the ligand binding moiety is linked (e.g.,covalently) to the C-terminal amino acid residue of the immunoglobulinchain. In certain embodiments, the ligand binding moiety is linked tothe C-terminal amino acid residue of an immunoglobulin chain without alinker (e.g., via a peptide bond). In certain embodiments, the ligandbinding moiety is linked (e.g., covalently) to the C-terminal amino acidresidue of the immunoglobulin chain via a linker. In certainembodiments, the linker is a peptide linker. In certain embodiments, thepeptide linker has the general formula (G₄S)_(n), where n is an integer.In certain embodiments, the peptide linker comprises an amino acidsequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 103, 104, 105,106, 107, or 108. In certain embodiments, the peptide linker consists ofan amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:103, 104, 105, 106, 107, or 108. In certain embodiments, the peptidelinker is linked to the immunoglobulin chain via a peptide bond.

In certain embodiments, the ligand binding moiety is linked (e.g.,covalently) to a VH of the anti-CD73 antibody. The ligand binding moietycan be linked (e.g., covalently) to any portion of the VH, including theN- or C-terminal amino acid residue. In certain embodiments, the ligandbinding moiety is linked (e.g., covalently) to the C-terminal amino acidresidue of the VH. In certain embodiments, the ligand binding moiety islinked to the C-terminal amino acid residue of the VH without a linker(e.g., via a peptide bond). In certain embodiments, the ligand bindingmoiety is linked (e.g., covalently) to the C-terminal amino acid residueof the VH via linker. In certain embodiments, the linker is a peptidelinker. In certain embodiments, the peptide linker comprises an aminoacid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 103, 104,105, 106, 107, or 108. In certain embodiments, the peptide linkerconsists of an amino acid sequence with at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto SEQ ID NO: 103, 104, 105, 106, 107, or 108. In certain embodiments,the peptide linker is linked to the VH via a peptide bond.

In certain embodiments, the ligand binding moiety is linked (e.g.,covalently) to a VL of the anti-CD73 antibody. The ligand binding moietycan be linked (e.g., covalently) to any portion of the VL, including theN- or C-terminal amino acid residue. In certain embodiments, the ligandbinding moiety is linked (e.g., covalently) to the C-terminal amino acidresidue of the VL. In certain embodiments, the ligand binding moiety islinked to the C-terminal amino acid residue of the VL without a linker(e.g., via a peptide bond). In certain embodiments, the ligand bindingmoiety is linked (e.g., covalently) to the C-terminal amino acid residueof the VL via linker. In certain embodiments, the linker is a peptidelinker. In certain embodiments, the peptide linker comprises an aminoacid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 103, 104,105, 106, 107, or 108. In certain embodiments, the peptide linkerconsists of an amino acid sequence with at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto SEQ ID NO: 103, 104, 105, 106, 107, or 108. In certain embodiments,the peptide linker is linked to the VL via a peptide bond.

In certain embodiments, the ligand binding moiety is linked (e.g.,covalently) to a heavy chain constant region of the anti-CD73 antibody.The ligand binding moiety can be linked (e.g., covalently) to anyportion of the heavy chain constant region. In certain embodiments, theligand binding moiety is linked (e.g., covalently) to the C-terminalamino acid residue of the heavy chain constant region. In certainembodiments, the ligand binding moiety is linked to the C-terminal aminoacid residue of the heavy chain constant region without a linker (e.g.,via a peptide bond). In certain embodiments, the ligand binding moietyis linked (e.g., covalently) to the C-terminal amino acid residue of theheavy chain constant region via linker. In certain embodiments, thelinker is a peptide linker. In certain embodiments, the peptide linkercomprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 103, 104, 105, 106, 107, or 108. In certain embodiments, thepeptide linker consists of an amino acid sequence with at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO: 103, 104, 105, 106, 107, or 108. In certainembodiments, the peptide linker is linked to the heavy chain constantregion via a peptide bond.

In certain embodiments, the ligand binding moiety is linked (e.g.,covalently) to a light chain constant region of the anti-CD73 antibody.The ligand binding moiety can be linked (e.g., covalently) to anyportion of the light chain constant region. In certain embodiments, theligand binding moiety is linked (e.g., covalently) to the C-terminalamino acid residue of the light chain constant region. In certainembodiments, the ligand binding moiety is linked to the C-terminal aminoacid residue of the light chain constant region without a linker (e.g.,via a peptide bond). In certain embodiments, the ligand binding moietyis linked (e.g., covalently) to the C-terminal amino acid residue of thelight chain constant region via linker. In certain embodiments, thelinker is a peptide linker. In certain embodiments, the peptide linkercomprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQID NO: 103, 104, 105, 106, 107, or 108. In certain embodiments, thepeptide linker consists of an amino acid sequence with at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO: 103, 104, 105, 106, 107, or 108. In certainembodiments, the peptide linker is linked to the light chain constantregion via a peptide bond.

The ligand binding moiety can be any moiety that has the ability tospecifically bind to a target molecule or complex. In certainembodiments, the ligand binding moiety comprises a peptide orpolypeptide. In certain embodiments, the peptide or polypeptide islinked to the immunoglobulin chain of the anti-CD73 antibody through apeptide bond. In certain embodiments, the peptide or polypeptide islinked to the immunoglobulin chain of the anti-CD73 antibody via apeptide linker, wherein peptide or polypeptide is linked to the peptidelinker through a peptide bond. In certain embodiments, the N-terminalamino acid residue of the peptide or polypeptide is linked to theC-terminal amino acid residue of the immunoglobulin chain or the peptidelinker through a peptide bond.

In certain embodiments, the anti-CD73 antibody comprises a polypeptidecomprising (a) an immunoglobulin chain, (b) a ligand binding moiety, andoptionally (c) a peptide linker as disclosed herein. In certainembodiments, the anti-CD73 antibody comprises a polypeptide comprisingfrom N-terminus to C-terminus the immunoglobulin chain and the bindingmoiety. In certain embodiments, the anti-CD73 antibody comprises apolypeptide comprising from N-terminus to C-terminus: the immunoglobulinchain, the peptide linker, and the binding moiety. In certainembodiments, the anti-CD73 antibody consists of a polypeptide comprising(a) an immunoglobulin chain, (b) the ligand binding moiety, andoptionally (c) a peptide linker as disclosed herein. In certainembodiments, the anti-CD73 antibody consists of a polypeptide comprisingfrom N-terminus to C-terminus the immunoglobulin chain and the bindingmoiety. In certain embodiments, the anti-CD73 antibody consists of apolypeptide comprising from N-terminus to C-terminus: the immunoglobulinchain, the peptide linker, and the binding moiety. In certainembodiments, the immunoglobulin chain is a full-length heavy chain. Incertain embodiments, the immunoglobulin chain comprising is afull-length light chain.

In certain embodiments, the anti-CD73 antibody comprises a bindingmoiety capable of antagonizing and/or inhibiting TGFβ signaling pathway.In certain embodiments, the anti-CD73 antibody comprises a bindingmoiety capable of antagonizing and/or inhibiting TGFβ. In certainembodiments, the anti-CD73 antibody comprises a TGFβ-binding moiety. TheTGFβ-binding moiety can be any moiety that specifically binds to one ormore family members or isoforms of TGFβ. In certain embodiments, theTGFβ-binding moiety specifically binds to TGFβ1 (e.g., human TGFβ1). Incertain embodiments, the TGFβ-binding moiety specifically binds to TGFβ2(e.g., human TGFβ2). In certain embodiments, the TGFβ-binding moietyspecifically binds to TGFβ3 (e.g., human TGFβ3). In certain embodiments,the TGFβ-binding moiety specifically binds to at least two of TGFβ1(e.g., human TGFβ1), TGFβ2 (e.g., human TGFβ2), and TGFβ3 (e.g., humanTGFβ3). In certain embodiments, the TGFβ-binding moiety specificallybinds to TGFβ1 (e.g., human TGFβ1) and TGFβ3 (e.g., human TGFβ3). Incertain embodiments, the TGFβ-binding moiety specifically binds to TGFβ1(e.g., human TGFβ 1), TGFβ2 (e.g., human TGFβ2), and TGFβ3 (e.g., humanTGFβ3). The skilled worker will appreciate that a TGFβ-binding moietythat specifically binds to one family member or isoform of TGFβ may bindto one or more other family members or isoforms of TGFβ with similar orhigher affinity. Exemplary TGFβ-binding moieties are disclosed in DeCrescenzo et al. (2008) Transforming Growth Factor-β in Cancer Therapy,Volume II, Cancer Drug Discovery and Development, Humana Press;Zwaagstra et al. (2012) Mol Cancer Ther. 11(7):1477-87; Ravi et al.(2018) Nat. Commun. 9:741; EP0975771B1, U.S. Pat. No. 7,786,261B2, U.S.Pat. No. 8,993,524B2, and US20150225483A1, each of which is incorporatedherein by reference in its entirety.

In certain embodiments, the TGFβ-binding moiety comprises a domain of aprotein that binds to TGFβ1 (e.g., human TGFβ1), TGFβ2 (e.g., humanTGFβ2), and/or TGFβ3 (e.g., human TGFβ3), or a variant thereof that hassimilar or improved TGFβ binding affinity. In certain embodiments, thedomain is an extracellular domain of a TGFβ receptor (e.g., a human TGFβreceptor). In certain embodiments, the domain is a TGFβ-binding domainof a TGFβ receptor (e.g., a human TGFβ receptor). In certainembodiments, the TGFβ receptor is selected from the group consisting ofTGFβR1 (e.g., human TGFβR1), TGFβR2 (e.g., human TGFβR2), and TGFβR3(e.g., human TGFβR3). In certain embodiments, the TGFβ-binding moietycomprises an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thesequence set forth in SEQ ID NO: 109, 110, 111, or 112. In certainembodiments, the TGFβ-binding moiety comprises the amino acid sequenceset forth in SEQ ID NO: 109, 110, 111, or 112. In certain embodiments,the TGFβ-binding moiety consists of an amino acid sequence at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to the sequence set forth in SEQ ID NO: 109, 110, 111, or112. In certain embodiments, the TGFβ-binding moiety consists of theamino acid sequence set forth in SEQ ID NO: 109, 110, 111, or 112.

In certain embodiments, the anti-CD73 antibody comprises a polypeptidecomprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thesequence set forth in SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, 120,or 121. In certain embodiments, the anti-CD73 antibody comprises apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:113, 114, 115, 116, 117, 118, 119, 120, or 121. In certain embodiments,the anti-CD73 antibody comprises a polypeptide consisting of an aminoacid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth inSEQ ID NO: 113, 114, 115, 116, 117, 118, 119, 120, or 121. In certainembodiments, the anti-CD73 antibody comprises a polypeptide consistingof the amino acid sequence set forth in SEQ ID NO: 113, 114, 115, 116,117, 118, 119, 120, or 121.

In certain embodiments, the anti-CD73 antibody comprising a TGFβ-bindingmoiety further comprises a polypeptide comprising an amino acid sequenceat least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:97, 92, or 128. In certain embodiments, the anti-CD73 antibodycomprising a TGFβ-binding moiety further comprises a polypeptidecomprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thesequence set forth in SEQ ID NO: 97. In certain embodiments, theanti-CD73 antibody comprising a TGFβ-binding moiety further comprises apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:97. In certain embodiments, the anti-CD73 antibody comprising aTGFβ-binding moiety further comprises a polypeptide consisting of anamino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence setforth in SEQ ID NO: 97. In certain embodiments, the anti-CD73 antibodycomprising a TGFβ-binding moiety further comprises a polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO: 97.

In certain embodiments, the anti-CD73 antibody comprises a firstpolypeptide and a second polypeptide, wherein the first and secondpolypeptides comprise the amino acid sequences set forth in SEQ ID NOs:113 and 92, 114 and 97, 115 and 97, 116 and 97, 117 and 97, 118 and 97,119 and 97, 120 and 97, or 121 and 97, respectively. In certainembodiments, the first and second polypeptides consist of the amino acidsequences set forth in SEQ ID NOs: 113 and 92, 114 and 97, 115 and 97,116 and 97, 117 and 97, 118 and 97, 119 and 97, 120 and 97, or 121 and97, respectively. In certain embodiments, the anti-CD73 antibodycomprises two polypeptides each comprising the amino acid sequence ofthe first polypeptide, and two polypeptides each comprising the aminoacid sequence of the second polypeptide. In certain embodiments, theanti-CD73 antibody comprises two copies of the first polypeptide and twocopies of the second polypeptide. In certain embodiments, the anti-CD73antibody consists of two copies of the first polypeptide and two copiesof the second polypeptide.

In certain embodiments, the TGFβ-binding moiety comprises an antibodythat specifically binds to TGFβ1 (e.g., human TGFβ1), TGFβ2 (e.g., humanTGFβ2), and/or TGFβ3 (e.g., human TGFβ3), or an antigen-binding fragmentthereof Exemplary anti-TGFβ antibodies include fresolumimab andmetelimumab, and are described in U.S. Pat. No. 6,492,497B1, U.S. Pat.No. 7,151,169B2, U.S. Pat. No. 7,723,486B2, which are incorporatedherein by reference in their entirety.

In certain embodiments, the anti-CD73 antibody comprises a bindingmoiety capable of antagonizing and/or inhibiting a VEGF signalingpathway. In certain embodiments, the anti-CD73 antibody comprises abinding moiety capable of antagonizing and/or inhibiting VEGF. Incertain embodiments, the anti-CD73 antibody comprises a VEGF-bindingmoiety. The VEGF-binding moiety can be any moiety that specificallybinds to one or more family members or isoforms of VEGF. In certainembodiments, the VEGF-binding moiety specifically binds to VEGF-A (e.g.,human VEGF-A). In certain embodiments, the VEGF-binding moietyspecifically binds to VEGF-B (e.g., human VEGF-B). In certainembodiments, the VEGF-binding moiety specifically binds to VEGF-C (e.g.,human VEGF-C). In certain embodiments, the VEGF-binding moietyspecifically binds to VEGF-D (e.g., human VEGF-D). In certainembodiments, the VEGF-binding moiety specifically binds to at least twoof VEGF-A (e.g., human VEGF-A), VEGF-B (e.g., human VEGF-B), VEGF-C(e.g., human VEGF-C), and VEGF-D (e.g., human VEGF-D). In certainembodiments, the VEGF-binding moiety specifically binds to VEGF-A (e.g.,human VEGF-A) and VEGF-B (e.g., human VEGF-B). In certain embodiments,the VEGF-binding moiety specifically binds to VEGF-C (e.g., humanVEGF-C) and VEGF-D (e.g., human VEGF-D). In certain embodiments, theVEGF-binding moiety specifically binds to at least three of VEGF-A(e.g., human VEGF-A), VEGF-B (e.g., human VEGF-B), VEGF-C (e.g., humanVEGF-C), and VEGF-D (e.g., human VEGF-D). In certain embodiments, theVEGF-binding moiety specifically binds to VEGF-A (e.g., human VEGF-A),VEGF-B (e.g., human VEGF-B), VEGF-C (e.g., human VEGF-C), and VEGF-D(e.g., human VEGF-D). The skilled worker will appreciate that aVEGF-binding moiety that specifically binds to one family member orisoform of VEGF may bind to one or more other family members or isoformsof VEGF with similar or higher affinity. Exemplary VEGF-binding moietiesinclude aflibercept and are disclosed in Holash et al. (2002) PNAS 99(17): 11393-98; U.S. Pat. Nos. 7,306,799B2, 7,608,261B2; and7,531,173B2, each of which is incorporated herein by reference in itsentirety.

In certain embodiments, the VEGF-binding moiety comprises a domain of aprotein that binds to VEGF-A (e.g., human VEGF-A), VEGF-B (e.g., humanVEGF-B), VEGF-C (e.g., human VEGF-C), and/or VEGF-D (e.g., humanVEGF-D), or a variant thereof that has similar or improved VEGF bindingaffinity. In certain embodiments, the domain is an extracellular domainof a VEGF receptor (e.g., a human VEGF receptor). In certainembodiments, the domain is a VEGF-binding domain of a VEGF receptor(e.g., a human VEGF receptor). In certain embodiments, the VEGF-bindingdomain comprises one, two, three, four, five, six, or seven of theimmunoglobulin-like domains of a VEGF receptor (e.g., a human VEGFreceptor). In certain embodiments, the VEGF-binding domain comprises oneor more, two or more, three or more, four or more, five or more, or sixor more of the immunoglobulin-like domains of a VEGF receptor (e.g., ahuman VEGF receptor). In certain embodiments, the VEGF-binding domaincomprises at most two, three, four, five, or six of theimmunoglobulin-like domains of a VEGF receptor (e.g., a human VEGFreceptor). In certain embodiments, the VEGF receptor is selected fromthe group consisting of VEGFR1 (e.g., human VEGFR1), VEGFR2 (e.g., humanVEGFR2), and VEGFR3 (e.g., human VEGFR3). In certain embodiments, theVEGF-binding moiety comprises an extracellular domain of VEGFR1 (e.g.,human VEGFR1). In certain embodiments, the VEGF-binding moiety comprisesan amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence setforth in SEQ ID NO: 122. In certain embodiments, the VEGF-binding moietycomprises an amino acid sequence set forth in SEQ ID NO: 122. In certainembodiments, the VEGF-binding moiety consists of an amino acid sequenceat least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:122. In certain embodiments, the VEGF-binding moiety consists of anamino acid sequence set forth in SEQ ID NO: 122. In certain embodiments,the VEGF-binding moiety comprises an extracellular domain of VEGFR2(e.g., human VEGFR2). In certain embodiments, the VEGF-binding moietycomprises an extracellular domain of VEGFR3 (e.g., human VEGFR3).

In certain embodiments, the anti-CD73 antibody comprises a polypeptidecomprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thesequence set forth in SEQ ID NO: 123, 124, 125, 126, or 127. In certainembodiments, the anti-CD73 antibody comprises a polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 123, 124, 125, 126, or127. In certain embodiments, the anti-CD73 antibody comprises apolypeptide consisting of an amino acid sequence at least 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the sequence set forth in SEQ ID NO: 123, 124, 125, 126, or127. In certain embodiments, the anti-CD73 antibody comprises apolypeptide consisting of the amino acid sequence set forth in SEQ IDNO: 123, 124, 125, 126, or 127.

In certain embodiments, the anti-CD73 antibody comprising a VEGF-bindingmoiety further comprises a polypeptide comprising an amino acid sequenceat least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:97, 92, or 128. In certain embodiments, the anti-CD73 antibodycomprising a VEGF-binding moiety further comprises a polypeptidecomprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thesequence set forth in SEQ ID NO: 97. In certain embodiments, theanti-CD73 antibody comprising a VEGF-binding moiety further comprises apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:97. In certain embodiments, the anti-CD73 antibody comprising aVEGF-binding moiety further comprises a polypeptide consisting of anamino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence setforth in SEQ ID NO: 97. In certain embodiments, the anti-CD73 antibodycomprising a VEGF-binding moiety further comprises a polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO: 97.

In certain embodiments, the anti-CD73 antibody comprises a firstpolypeptide and a second polypeptide, wherein the first and secondpolypeptides comprise the amino acid sequences set forth in SEQ ID NOs:123 and 92, 124 and 128, 125 and 128, 126 and 97, or 127 and 97,respectively. In certain embodiments, the first and second polypeptidesconsist of the amino acid sequences set forth in SEQ ID NOs: 123 and 92,124 and 128, 125 and 128, 126 and 97, or 127 and 97, respectively. Incertain embodiments, the anti-CD73 antibody comprises two polypeptideseach comprising the amino acid sequence of the first polypeptide, andtwo polypeptides comprising the amino acid sequence of the secondpolypeptide. In certain embodiments, the anti-CD73 antibody comprisestwo copies of the first polypeptide and two copies of the secondpolypeptide. In certain embodiments, the anti-CD73 antibody consists oftwo copies of the first polypeptide and two copies of the secondpolypeptide. In certain embodiments, the VEGF-binding moiety comprisesan antibody that specifically binds to VEGF-A (e.g., human VEGF-A),VEGF-B (e.g., human VEGF-B), VEGF-C (e.g., human VEGF-C), and/or VEGF-D(e.g., human VEGF-D), or an antigen-binding fragment thereof. Exemplaryanti-VEGF antibodies include bevacizumab and ranibizumab, and aredescribed in WO1998045331A2 and WO1998045332A2, which are incorporatedherein by reference in their entirety.

5.4 Pharmaceutical Compositions

Provided herein are compositions comprising an anti-CD73 (e.g., human,mouse, or cynomolgus CD73) antibody disclosed herein having the desireddegree of purity in a physiologically acceptable carrier, excipient orstabilizer (see, e.g., Remington's Pharmaceutical Sciences (1990) MackPublishing Co., Easton, PA). Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

In a specific embodiment, pharmaceutical compositions comprise ananti-CD73 (e.g., human. Mouse, or cynomolgus CD73) antibody disclosedherein, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In aspecific embodiment, pharmaceutical compositions comprise an effectiveamount of an antibody described herein, and optionally one or moreadditional prophylactic or therapeutic agents, in a pharmaceuticallyacceptable carrier. In certain embodiments, the antibody is the onlyactive ingredient included in the pharmaceutical composition.Pharmaceutical compositions described herein can be useful in decreasingor inhibiting CD73 (e.g., human, mouse, or cynomolgus CD73) activity andtreating a condition, such as cancer or an infectious disease. In oneembodiment, the present invention relates to a pharmaceuticalcomposition of the present invention comprising an anti-CD73 antibody ofthe present invention for use as a medicament. In another embodiment,the present invention relates to a pharmaceutical composition of thepresent invention for use in a method for the treatment of cancer or aninfectious disease.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parenteral. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody includesterile solutions ready for injection, sterile dry soluble products,such as lyophilized powders, ready to be combined with a solvent justprior to use, including hypodermic tablets, sterile suspensions readyfor injection, sterile dry insoluble products ready to be combined witha vehicle just prior to use and sterile emulsions. The solutions may beeither aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising antibody proteins are prepared as describedfor the local and systemic administration. The resulting mixture can bea solution, suspension, emulsions or the like and can be formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

An anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody disclosedherein can be formulated as an aerosol for topical application, such asby inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209 and4,364,923, which describe aerosols for delivery of a steroid useful fortreatment of inflammatory diseases, particularly asthma and are hereinincorporated by reference in their entireties). These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflations,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, in one embodiment, havediameters of less than 50 microns, in one embodiment less than 10microns.

An anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody disclosedherein can be formulated for local or topical application, such as fortopical application to the skin and mucous membranes, such as in theeye, in the form of gels, creams, and lotions and for application to theeye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the antibody alone or in combination with otherpharmaceutically acceptable excipients can also be administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody. For example, such patches are disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of whichare herein incorporated by reference in their entireties.

In certain embodiments, a pharmaceutical composition comprising anantibody described herein is a lyophilized powder, which can bereconstituted for administration as solutions, emulsions and othermixtures. It may also be reconstituted and formulated as solids or gels.The lyophilized powder is prepared by dissolving an antibody describedherein, or a pharmaceutically acceptable derivative thereof, in asuitable solvent. In certain embodiments, the lyophilized powder issterile. The solvent may contain an excipient which improves thestability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

The anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibodiesdisclosed herein and other compositions provided herein can also beformulated to be targeted to a particular tissue, receptor, or otherarea of the body of the subject to be treated. Many such targetingmethods are well known to those of skill in the art. All such targetingmethods are contemplated herein for use in the instant compositions. Fornon-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos.6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570,6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534,5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874, allof which are herein incorporated by reference in their entireties. In aspecific embodiment, an antibody described herein is targeted to atumor.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

5.5 Methods of Use and Uses

In another aspect, the instant disclosure provides a method of treatinga subject using the anti-CD73 (e.g., human, mouse, or cynomolgus CD73)antibodies disclosed herein. Any disease or disorder in a subject thatwould benefit from decrease of CD73 (e.g., human, mouse, or cynomolgusCD73) function and/or signaling related to TGFβ, VEGF, and/or adenosinecan be treated using the anti-CD73 (e.g., human, mouse or cynomolgusCD73) antibodies disclosed herein. In certain embodiments, the diseaseor disorder is resistant to an immunotherapy, such as a checkpointtargeting agent (e.g., an antagonist anti-CTLA-4 antibody, an antagonistanti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, an antagonistanti-PD-1 antibody, an antagonist anti-TIM-3 antibody, an antagonistanti-LAG-3 antibody, an antagonist anti-CEACAM1 antibody, an agonistanti-GITR antibody, an antagonist anti-TIGIT antibody, an antagonistanti-VISTA antibody, an agonist anti-CD137 antibody, or an agonistanti-OX40 antibody), a TCR T cell therapy, or a chimeric antigenreceptor (CAR) T cell therapy (e.g., a CD19 CAR T cell therapy). Incertain embodiments, the disease or disorder is recurrent aftertreatment with an immunotherapy, such as a checkpoint targeting agent(e.g., an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1antibody, an antagonist anti-PD-L2 antibody, an antagonist anti-PD-1antibody, an antagonist anti-TIM-3 antibody, an antagonist anti-LAG-3antibody, an antagonist anti-CEACAM1 antibody, an agonist anti-GITRantibody, an antagonist anti-TIGIT antibody, an antagonist anti-VISTAantibody, an agonist anti-CD137 antibody, or an agonist anti-OX40antibody), a TCR T cell therapy, or a CAR T cell therapy. In certainembodiments, the disease or disorder is resistant to a chemotherapy(e.g., a platinum-based therapy (e.g., cisplatin, carboplatin, oroxaliplatin), a tyrosine kinase (e.g., VEGFR, Raf) inhibitor (e.g.,sorafenib), a DNA damage-inducing agent (e.g., gemcitabine), ahypomethylating agent (e.g., azacitidine), and/or a combination ofmultiple chemotherapies (e.g., a platinum doublet or a FOLFOX regimen(e.g., FOLFIRINOX or mFOLFOX6))). In certain embodiments, the disease ordisorder is recurrent after treatment with a chemotherapy (e.g., aplatinum-based therapy (e.g., cisplatin, carboplatin, or oxaliplatin), atyrosine kinase (e.g., VEGFR, Raf) inhibitor (e.g., sorafenib), a DNAdamage-inducing agent (e.g., gemcitabine), a hypomethylating agent(e.g., azacitidine), and/or a combination of multiple chemotherapies(e.g., a platinum doublet or a FOLFOX regimen (e.g., FOLFIRINOX ormFOLFOX6))). In certain embodiments, the disease or disorder isresistant to an anti-angiogenic therapy (e.g., bevacizumab). In certainembodiments, the disease or disorder is recurrent after treatment withan anti-angiogenic therapy (e.g., bevacizumab). In certain embodiments,the solid tumor is a measurable disease.

The anti-CD73 (e.g., human CD73) antibodies disclosed herein areparticularly useful for inhibiting immune system tolerance to tumors,and accordingly can be used as an immunotherapy for subjects withcancer. For example, in certain embodiments, the instant disclosureprovides a method of treating cancer in a subject, the method comprisingadministering to the subject an effective amount of the antibody,polynucleotide, vector, lipid nanoparticle, or pharmaceuticalcomposition, as disclosed herein.

Cancers that can be treated with the anti-CD73 (e.g., human, mouse, orcynomolgus CD73) antibodies, or pharmaceutical compositions disclosedherein include, without limitation, a solid tumor, a hematologicalcancer (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), anda metastatic lesion. In one embodiment, the cancer is a solid tumor.Examples of solid tumors include malignancies, e.g., sarcomas,fibroblastic sarcoma, and carcinomas, e.g., adenocarcinomas of thevarious organ systems, such as those affecting the lung, breast,ovarian, lymphoid, gastrointestinal (e.g., colon), anal, genitals andgenitourinary tract (e.g., renal, urothelial, bladder cells, prostate),pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin(e.g., melanoma), and pancreas, as well as adenocarcinomas which includemalignancies such as colon cancers, rectal cancer, renal-cell carcinoma,liver cancer, lung cancer (e.g., non-small cell lung cancer or smallcell lung cancer), cancer of the small intestine and cancer of theesophagus. The cancer may be at an early, intermediate, late stage ormetastatic cancer. In certain embodiments, the cancer is resistant to animmunotherapy, such as a checkpoint targeting agent (e.g., an antagonistanti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonistanti-PD-L2 antibody, or an antagonist anti-PD-1 antibody). In certainembodiments, the cancer is recurrent after treatment with animmunotherapy, such as a checkpoint targeting agent (e.g., an antagonistanti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonistanti-PD-L2 antibody, an antagonist anti-PD-1 antibody, an antagonistanti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an antagonistanti-CEACAM1 antibody, an agonist anti-GITR antibody, an antagonistanti-TIGIT antibody, an antagonist anti-VISTA antibody, an agonistanti-CD137 antibody, or an agonist anti-OX40 antibody).

In one embodiment, the cancer is chosen from lung cancer (e.g., lungadenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC withsquamous and/or non-squamous histology, or NSCLC adenocarcinoma)),melanoma (e.g., an advanced melanoma), renal cancer (e.g., a renal cellcarcinoma), liver cancer (e.g., hepatocellular carcinoma), myeloma(e.g., a multiple myeloma), a prostate cancer (e.g.,castration-resistant prostate cancer), a breast cancer (e.g., a breastcancer that does not express one, two or all of estrogen receptor,progesterone receptor, or Her2/neu, e.g., a triple negative breastcancer), an ovarian cancer, a colorectal cancer, a pancreatic cancer, ahead and neck cancer (e.g., head and neck squamous cell carcinoma(HNSCC), glioblastoma (GBM), meningioma, endometrial cancer, gastric orstomach cancer, adrenal cancer, anal cancer, gastro-esophageal cancer(e.g., esophageal squamous cell carcinoma), mesothelioma, nasopharyngealcancer, thyroid cancer, cervical cancer, epithelial cancer, peritonealcancer, or a lymphoproliferative disease (e.g., a post-transplantlymphoproliferative disease). In a specific embodiment, the cancer is acervical cancer.

In one embodiment, the cancer is a hematological cancer, for example, aleukemia, a lymphoma, or a myeloma. In one embodiment, the cancer is aleukemia, for example, acute lymphoblastic leukemia (ALL) (e.g., non-Bcell ALL), acute myelogenous leukemia (AML), chronic lymphocyticleukemia (CLL), chronic myelogenous leukemia (CML), chronicmyelomonocytic leukemia (CMML), or hairy cell leukemia. In oneembodiment, the cancer is a lymphoma, for example, B cell lymphoma,diffuse large B-cell lymphoma (DLBCL), activated B-cell like (ABC)diffuse large B cell lymphoma, germinal center B cell (GCB) diffuselarge B cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma,non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractorynon-Hodgkin lymphoma, recurrent follicular non-Hodgkin lymphoma, Burkittlymphoma, small lymphocytic lymphoma, follicular lymphoma,lymphoplasmacytic lymphoma, extranodal marginal zone lymphoma, orminimal residual disease. In one embodiment the cancer is a myeloma, forexample, multiple myeloma.

In another embodiment, the cancer is chosen from a carcinoma (e.g.,advanced or metastatic carcinoma), melanoma or a lung carcinoma, e.g., anon-small cell lung carcinoma.

In one embodiment, the cancer is a lung cancer, e.g., a lungadenocarcinoma, non-small cell lung cancer, or small cell lung cancer.

In one embodiment, the cancer is a melanoma, e.g., an advanced melanoma.In one embodiment, the cancer is an advanced or unresectable melanomathat does not respond to other therapies. In other embodiments, thecancer is a melanoma with a BRAF mutation (e.g., a BRAF V600 mutation).In yet other embodiments, the anti-CD73 (e.g., human, mouse, orcynomolgus CD73) antibody or pharmaceutical composition disclosedherein, is administered after treatment with an anti-CTLA-4 antibody(e.g., ipilimumab) with or without a BRAF inhibitor (e.g., vemurafenibor dabrafenib).

In another embodiment, the cancer is a hepatocarcinoma, e.g., anadvanced hepatocarcinoma, with or without a viral infection, e.g., achronic viral hepatitis.

In another embodiment, the cancer is a prostate cancer, e.g., anadvanced prostate cancer.

In yet another embodiment, the cancer is a myeloma, e.g., multiplemyeloma.

In yet another embodiment, the cancer is a renal cancer, e.g., a renalcell carcinoma (RCC) (e.g., a metastatic RCC, clear cell renal cellcarcinoma (CCRCC) or kidney papillary cell carcinoma).

In yet another embodiment, the cancer is chosen from a lung cancer, amelanoma, a renal cancer, a breast cancer, a colorectal cancer, aleukemia, or a metastatic lesion of the cancer.

In certain embodiments, the instant disclosure provides a method ofpreventing or treating an infectious disease in a subject, the methodcomprising administering to the subject an effective amount of ananti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody orpharmaceutical composition thereof, as disclosed herein. In oneembodiment, provided herein are methods for preventing and/or treatingan infection (e.g., a viral infection, a bacterial infection, a fungalinfection, a protozoal infection, or a parasitic infection). Theinfection prevented and/or treated in accordance with the methods can becaused by an infectious agent identified herein. In a specificembodiment, an anti-CD73 (e.g., human, mouse, or cynomolgus CD73)antibody described herein or a composition thereof is the only activeagent administered to a subject. In certain embodiments, an anti-CD73(e.g., human, mouse, or cynomolgus CD73) antibody described herein or acomposition thereof is used in combination with anti-infectiveinterventions (e.g., antivirals, antibacterials, antifungals, oranti-helminthics) for the treatment of infectious diseases. Therefore,in a one embodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention for use in a methodof preventing and/or treating an infectious disease, optionally whereinthe antibody or pharmaceutical composition is the only active agentadministered to a subject, or wherein the antibody or pharmaceuticalcomposition is used in combination with anti-infective interventions.

Infectious diseases that can be treated and/or prevented by anti-CD73(e.g., human, mouse, or cynomolgus CD73) antibodies or pharmaceuticalcompositions disclosed herein are caused by infectious agents includingbut not limited to bacteria, parasites, fungi, protozoa, and viruses. Ina specific embodiment, the infectious disease treated and/or preventedby anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibodies orpharmaceutical compositions disclosed herein is caused by a virus. Viraldiseases or viral infections that can be prevented and/or treated inaccordance with the methods described herein include, but are notlimited to, those caused by hepatitis type A, hepatitis type B,hepatitis type C, influenza (e.g., influenza A or influenza B),varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, small pox,Epstein Barr virus, human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), and agents of viral diseasessuch as viral meningitis, encephalitis, dengue or small pox.

Bacterial infections that can be prevented and/or treated includeinfections caused by Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseasescaused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa) that can beprevented and/or treated in accordance with the methods described hereininclude, but are not limited to, Mycobacteria rickettsia, Mycoplasma,Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillusantracis (anthrax), tetanus, Streptococcus, Staphylococcus,mycobacterium, pertissus, cholera, plague, diptheria, chlamydia, S.aureus and legionella.

Protozoal diseases or protozoal infections caused by protozoa that canbe prevented and/or treated in accordance with the methods describedherein include, but are not limited to, leishmania, coccidiosis,Trypanosoma schistosoma or malaria. Parasitic diseases or parasiticinfections caused by parasites that can be prevented and/or treated inaccordance with the methods described herein include, but are notlimited to, chlamydia and rickettsia.

Fungal diseases or fungal infections that can be prevented and/ortreated in accordance with the methods described herein include, but arenot limited to, those caused by Candida infections, zygomycosis, Candidamastitis, progressive disseminated trichosporonosis with latenttrichosporonemia, disseminated candidiasis, pulmonaryparacoccidioidomycosis, pulmonary aspergillosis, Pneumocystis cariniipneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis andcerebrospinal vasculitis, Aspergillus niger infection, Fusariumkeratitis, paranasal sinus mycoses, Aspergillus fumigatus endocarditis,tibial dyschondroplasia, Candida glabrata vaginitis, oropharyngealcandidiasis, X-linked chronic granulomatous disease, tinea pedis,cutaneous candidiasis, mycotic placentitis, disseminatedtrichosporonosis, allergic bronchopulmonary aspergillosis, mycotickeratitis, Cryptococcus neoformans infection, fungal peritonitis,Curvularia geniculata infection, staphylococcal endophthalmitis,sporotrichosis, and dermatophytosis.

In certain embodiments, the anti-CD73 (e.g., human, mouse, or cynomolgusCD73) antibodies or pharmaceutical compositions disclosed herein areadministered to the subject as a monotherapy.

In certain embodiments, these methods further comprise administering anadditional therapeutic agent to the subject. In certain embodiments, theadditional therapeutic agent comprises a chemotherapeutic agent. Incertain embodiments, the chemotherapeutic agent comprises ahypomethylating agent (e.g., azacitidine). In certain embodiments, thechemotherapeutic agent comprises a DNA damage-inducing agent (e.g.,gemcitabine). In certain embodiments, the chemotherapeutic agentcomprises a platinum-based therapy (e.g., cisplatin, carboplatin, oroxaliplatin). In certain embodiments, the chemotherapeutic agentcomprises a tyrosine kinase (e.g., VEGFR, Raf) inhibitor (e.g.,sorafenib). In certain embodiments, the chemotherapeutic agent comprisesa combination of multiple chemotherapies (e.g., a platinum doublet or aFOLFOX regimen (e.g., FOLFIRINOX or mFOLFOX6)). In certain embodiments,the additional therapeutic agent comprises an anti-angiogenic therapy(e.g., bevacizumab). In certain embodiments, the additional therapeuticagent comprises an antibody-drug conjugate. In certain embodiments, theadditional therapeutic agent comprises a radiotherapeutic agent. Incertain embodiments, the additional therapeutic agent is capable ofcausing immunogenic cell death (e.g., tumor cell death).

In certain embodiments, the additional therapeutic agent comprises acheckpoint targeting agent. In certain embodiments, the checkpointtargeting agent is selected from the group consisting of an antagonistanti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonistanti-PD-L2 antibody, an antagonist anti-PD-1 antibody, an antagonistanti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an antagonistanti-TIGIT antibody, an antagonist anti-VISTA antibody, an antagonistanti-CD96 antibody, an antagonist anti-CEACAM1 antibody, an agonistanti-CD137 antibody, an agonist anti-GITR antibody, and an agonistanti-OX40 antibody. In certain embodiments, the checkpoint targetingagent is selected from the group consisting of an antagonist anti-CTLA-4antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2antibody, and an antagonist anti-PD-1 antibody, wherein the anti-CD73(e.g., human, mouse, or cynomolgus CD73) antibodies or pharmaceuticalcompositions disclosed herein synergize with the checkpoint targetingagent.

In one embodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention for use in a methodof the present invention, wherein the method further comprisesadministering an additional therapeutic agent to the subject. In oneembodiment, the present invention relates to (a) an antibody and/orpharmaceutical composition of the present invention and (b) anadditional therapeutic agent for use as a medicament. In one embodiment,the present invention relates to (a) an antibody and/or pharmaceuticalcomposition of the present invention, and (b) an additional therapeuticagent for use in a method for the treatment of cancer. In a furtherembodiment, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody and/orpharmaceutical composition of the present invention and (b) anadditional therapeutic agent. In one embodiment, the additionaltherapeutic agent is a chemotherapeutic, a radiotherapeutic, or acheckpoint targeting agent.

In certain embodiments, an anti-PD-1 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-PD-1 antibody isnivolumab, also known as BMS-936558 or MDX1106, developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody ispembrolizumab, also known as lambrolizumab or MK-3475, developed byMerck & Co. In certain embodiments, the anti-PD-1 antibody ispidilizumab, also known as CT-011, developed by CureTech. In certainembodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514,developed by Medimmune. In certain embodiments, the anti-PD-1 antibodyis PDR001 developed by Novartis Pharmaceuticals. In certain embodiments,the anti-PD-1 antibody is REGN2810 developed by RegeneronPharmaceuticals. In certain embodiments, the anti-PD-1 antibody isPF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1antibody is BGB-A317 developed by BeiGene. In certain embodiments, theanti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. Incertain embodiments, the anti-PD-1 antibody is SHR-1210 developed byHengrui.

Further non-limiting examples of anti-PD-1 antibodies that may be usedin treatment methods disclosed herein are disclosed in the followingpatents and patent applications, all of which are herein incorporated byreference in their entireties for all purposes: U.S. Pat. Nos.6,808,710; 7,332,582; 7,488,802; 8,008,449; 8,114,845; 8,168,757;8,354,509; 8,686,119; 8,735,553; 8,747,847; 8,779,105; 8,927,697;8,993,731; 9,102,727; 9,205,148; U.S. Publication No. US 2013/0202623A1; U.S. Publication No. US 2013/0291136 A1; U.S. Publication No. US2014/0044738 A1; U.S. Publication No. US 2014/0356363 A1; U.S.Publication No. US 2016/0075783 A1; and PCT Publication No. WO2013/033091 A1; PCT Publication No. WO 2015/036394 A1; PCT PublicationNo. WO 2014/179664 A2; PCT Publication No. WO 2014/209804 A1; PCTPublication No. WO 2014/206107 A1; PCT Publication No. WO 2015/058573A1; PCT Publication No. WO 2015/085847 A1; PCT Publication No. WO2015/200119 A1; PCT Publication No. WO 2016/015685 A1; and PCTPublication No. WO 2016/020856 A1.

In certain embodiments, an anti-PD-L1 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-PD-L1 antibody isatezolizumab developed by Genentech. In certain embodiments, theanti-PD-L1 antibody is durvalumab developed by AstraZeneca, Celgene andMedimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab,also known as MSB0010718C, developed by Merck Serono and Pfizer. Incertain embodiments, the anti-PD-L1 antibody is MDX-1105 developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody isAMP-224 developed by Amplimmune and GSK.

Non-limiting examples of anti-PD-L1 antibodies that may be used intreatment methods disclosed herein are disclosed in the followingpatents and patent applications, all of which are herein incorporated byreference in their entireties for all purposes: U.S. Pat. Nos.7,943,743; 8,168,179; 8,217,149; 8,552,154; 8,779,108; 8,981,063;9,175,082; U.S. Publication No. US 2010/0203056 A1; U.S. Publication No.US 2003/0232323 A1; U.S. Publication No. US 2013/0323249 A1; U.S.Publication No. US 2014/0341917 A1; U.S. Publication No. US 2014/0044738A1; U.S. Publication No. US 2015/0203580 A1; U.S. Publication No. US2015/0225483 A1; U.S. Publication No. US 2015/0346208 A1; U.S.Publication No. US 2015/0355184 A1; and PCT Publication No. WO2014/100079 A1; PCT Publication No. WO 2014/022758 A1; PCT PublicationNo. WO 2014/055897 A2; PCT Publication No. WO 2015/061668 A1; PCTPublication No. WO 2015/109124 A1; PCT Publication No. WO 2015/195163A1; PCT Publication No. WO 2016/000619 A1; and PCT Publication No. WO2016/030350 A1.

In certain embodiments, an anti-CTLA-4 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-CTLA-4 antibody isipilimumab developed by Bristol-Myers Squibb.

In certain embodiments, an anti-CD73 (e.g., human, mouse, or cynomolgusCD73) antibody disclosed herein is administered to a subject incombination with a compound that targets an immunomodulatory enzyme(s)such as IDO (indoleamine-(2,3)-dioxygenase) and/or TDO (tryptophan2,3-dioxygenase). Therefore, in one embodiment, the additionaltherapeutic agent is a compound that targets an immunomodulatoryenzyme(s), such as an inhibitor of indoleamine-(2,3)-dioxygenase (IDO).In certain embodiments, such compound is selected from the groupconsisting of epacadostat (Incyte Corp; see, e.g., WO 2010/005958 whichis herein incorporated by reference in its entirety), F001287 (FlexusBiosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), andNLG919 (NewLink Genetics). In one embodiment, the compound isepacadostat. In another embodiment, the compound is F001287. In anotherembodiment, the compound is indoximod. In another embodiment, thecompound is NLG919. In a specific embodiment, an anti-CD73 (e.g., humanCD73) antibody disclosed herein is administered to a subject incombination with an IDO inhibitor for treating cancer. The IDO inhibitoras described herein for use in treating cancer is present in a soliddosage form of a pharmaceutical composition such as a tablet, a pill ora capsule, wherein the pharmaceutical composition includes an IDOinhibitor and a pharmaceutically acceptable excipient. As such, theantibody as described herein and the IDO inhibitor as described hereincan be administered separately, sequentially or concurrently as separatedosage forms. In one embodiment, the antibody is administeredparenterally, and the IDO inhibitor is administered orally. Inparticular embodiments, the inhibitor is selected from the groupconsisting of epacadostat (Incyte Corporation), F001287 (FlexusBiosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), andNLG919 (NewLink Genetics). Epacadostat has been described in PCTPublication No. WO 2010/005958, which is herein incorporated byreference in its entirety for all purposes. In one embodiment, theinhibitor is epacadostat. In another embodiment, the inhibitor isF001287. In another embodiment, the inhibitor is indoximod. In anotherembodiment, the inhibitor is NLG919.

In certain embodiments, an anti-CD73 (e.g., human, mouse, or cynomolgusCD73) antibody disclosed herein is administered to a subject incombination with a vaccine. The vaccine can be, e.g., a peptide vaccine,a DNA vaccine, or an RNA vaccine. In certain embodiments, the vaccine isa heat shock protein-based tumor vaccine or a heat shock protein-basedpathogen vaccine. In a specific embodiment, an anti-CD73 (e.g., human,mouse, or cynomolgus CD73) antibody disclosed herein is administered toa subject in combination with a heat shock protein-based tumor-vaccine.Heat shock proteins (HSPs) are a family of highly conserved proteinsfound ubiquitously across all species. Their expression can bepowerfully induced to much higher levels as a result of heat shock orother forms of stress, including exposure to toxins, oxidative stress orglucose deprivation. Five families have been classified according tomolecular weight: HSP-110, -90, -70, -60 and -28. HSPs deliverimmunogenic peptides through the cross-presentation pathway in antigenpresenting cells (APCs) such as macrophages and dendritic cells (DCs),leading to T cell activation. HSPs function as chaperone carriers oftumor-associated antigenic peptides forming complexes able to inducetumor-specific immunity. Upon release from dying tumor cells, theHSP-antigen complexes are taken up by antigen-presenting cells (APCs)wherein the antigens are processed into peptides that bind MHC class Iand class II molecules leading to the activation of anti-tumor CD8+ andCD4+ T cells. The immunity elicited by HSP complexes derived from tumorpreparations is specifically directed against the unique antigenicpeptide repertoire expressed by the cancer of each subject. Therefore,in one embodiment, the present invention relates to (a) an antibodyand/or pharmaceutical composition of the present invention and (b) avaccine for use as a medicament, for example for use in a method for thetreatment of cancer. In one embodiment, the present invention relates toa pharmaceutical composition, kit or kit-of-parts comprising (a) anantibody and/or pharmaceutical composition of the present invention and(b) a vaccine. In one embodiment, the vaccine is a heat shockprotein-based tumor vaccine. In one embodiment, the vaccine is a heatshock protein-based pathogen vaccine. In certain embodiments, thevaccine is as described in WO 2016/183486, incorporated herein byreference in its entirety.

A heat shock protein peptide complex (HSPPC) is a protein peptidecomplex consisting of a heat shock protein non-covalently complexed withantigenic peptides. HSPPCs elicit both innate and adaptive immuneresponses. In a specific embodiment, the antigenic peptide(s) displaysantigenicity for the cancer being treated. HSPPCs are efficiently seizedby APCs via membrane receptors (mainly CD91) or by binding to Toll-likereceptors. HSPPC internalization results in functional maturation of theAPCs with chemokine and cytokine production leading to activation ofnatural killer cells (NK), monocytes and Th1 and Th-2-mediated immuneresponses. In certain embodiments, HSPPCs used in methods disclosedherein comprise one or more heat shock proteins from the hsp60, hsp70,or hsp90 family of stress proteins complexed with antigenic peptides. Incertain embodiments, HSPPCs comprise hsc70, hsp70, hsp90, hsp110,grp170, gp96, calreticulin, or combinations of two or more thereof.

In a specific embodiment, the heat shock protein peptide complex (HSPPC)comprises recombinant heat shock proteins (e.g., hsp70 or hsc70) or apeptide-binding domain thereof complexed with recombinant antigenicpeptides. Recombinant heat shock proteins can be produced by recombinantDNA technology, for example, using human hsc70 sequence as described inDworniczak and Mirault, Nucleic Acids Res. 15:5181-5197 (1987) andGenBank accession no. P11142 and/or Y00371, each of which isincorporated herein by reference in its entirety. In certainembodiments, Hsp70 sequences are as described in Hunt and Morimoto Proc.Natl. Acad. Sci. U.S.A. 82 (19), 6455-6459 (1985) and GenBank accessionno. PODMV8 and/or M11717, each of which is incorporated herein byreference in its entirety. Antigenic peptides can also be prepared byrecombinant DNA methods known in the art.

In certain embodiments, the antigenic peptides comprise a modified aminoacid. In certain embodiments, the modified amino acid comprises apost-translational modification. In certain embodiments, the modifiedamino acid comprises a mimetic of a post-translational modification. Incertain embodiments, the modified amino acid is a Tyr, Ser, Thr, Arg,Lys, or His that has been phosphorylated on a side chain hydroxyl oramine. In certain embodiments, the modified amino acid is a mimetic of aTyr, Ser, Thr, Arg, Lys, or His amino acid that has been phosphorylatedon a side chain hydroxyl or amine.

In a specific embodiment, an anti-CD73 (e.g., human, mouse, orcynomolgus CD73) antibody disclosed herein is administered to a subjectin combination with a heat shock protein peptide complex (HSPPC), e.g.,heat shock protein peptide complex-96 (HSPPC-96), to treat cancer.HSPPC-96 comprises a 96 kDa heat shock protein (Hsp), gp96, complexed toantigenic peptides. HSPPC-96 is a cancer immunotherapy manufactured froma subject's tumor and contains the cancer's antigenic “fingerprint.” Incertain embodiments, this fingerprint contains unique antigens that arepresent only in that particular subject's specific cancer cells andinjection of the vaccine is intended to stimulate the subject's immunesystem to recognize and attack any cells with the specific cancerfingerprint. Therefore, in one embodiment, the present invention relatesto an antibody and/or pharmaceutical composition of the presentinvention in combination with a heat shock protein peptide complex(HSPPC) for use as a medicament and/or for use in a method for thetreatment of cancer.

In certain embodiments, the HSPPC, e.g., HSPPC-96, is produced from thetumor tissue of a subject. In a specific embodiment, the HSPPC (e.g.,HSPPC-96) is produced from a tumor of the type of cancer or metastasisthereof being treated. In another specific embodiment, the HSPPC (e.g.,HSPPC-96) is autologous to the subject being treated. In certainembodiments, the tumor tissue is non-necrotic tumor tissue. In certainembodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 grams) of non-necrotic tumor tissue is used to produce avaccine regimen. In certain embodiments, after surgical resection,non-necrotic tumor tissue is frozen prior to use in vaccine preparation.In certain embodiments, the HSPPC, e.g., HSPPC-96, is isolated from thetumor tissue by purification techniques, filtered and prepared for aninjectable vaccine. In certain embodiments, a subject is administered6-12 doses of the HSPPC, e.g., HSPCC-96. In such embodiments, the HSPPC,e.g., HSPPC-96, doses may be administered weekly for the first 4 dosesand then biweekly for the 2-8 additional doses.

Further examples of HSPPCs that may be used in accordance with themethods described herein are disclosed in the following patents andpatent applications, all of which are herein incorporated by referencein their entireties: U.S. Pat. Nos. 6,391,306, 6,383,492, 6,403,095,6,410,026, 6,436,404, 6,447,780, 6,447,781 and 6,610,659. Examples ofHSPPCs include without limitation Prophage™, AutoSynVax™, andPhosphoSynVax™

In certain embodiments, an anti-CD73 (e.g., human, mouse, or cynomolgusCD73) antibody disclosed herein is administered to a subject incombination with an adjuvant. Various adjuvants can be used depending onthe treatment context. Non-limiting examples of appropriate adjuvantsinclude, but not limited to, Complete Freund's Adjuvant (CFA),Incomplete Freund's Adjuvant (IFA), montanide ISA (incomplete Seppicadjuvant), the Ribi adjuvant system (RAS), Titer Max, muramyl peptides,Syntex Adjuvant Formulation (SAF), alum (aluminum hydroxide and/oraluminum phosphate), aluminum salt adjuvants, Gerbu® adjuvants,nitrocellulose absorbed antigen, encapsulated or entrapped antigen, 3De-O-acylated monophosphoryl lipid A (3 D-MPL), immunostimulatoryoligonucleotides, toll-like receptor (TLR) ligands, mannan-bindinglectin (MBL) ligands, STING agonists, immuno-stimulating complexes suchas saponins, Quil A, QS-21, QS-7, ISCOMATRIX, and others. Otheradjuvants include CpG oligonucleotides and double stranded RNAmolecules, such as poly(A) and poly(U). Combinations of the aboveadjuvants may also be used. See, e.g., U.S. Pat. Nos. 6,645,495;7,029,678; and 7,858,589, all of which are incorporated herein byreference in their entireties. In one embodiment, the adjuvant usedherein is QS-21 STIMULON.

In certain embodiments, an anti-CD73 (e.g., human, mouse, or cynomolgusCD73) antibody disclosed herein is administered to a subject incombination with an additional therapeutic agent comprising a TCR. Incertain embodiments, the additional therapeutic agent is a soluble TCR.In certain embodiments, the additional therapeutic agent is a cellexpressing a TCR. Therefore, in one embodiment, the present inventionrelates to an antibody and/or pharmaceutical composition of the presentinvention in combination with an additional therapeutic agent comprisinga TCR for use as a medicament and/or for use in a method for thetreatment of cancer.

In certain embodiments, an anti-CD73 (e.g., human, mouse, or cynomolgusCD73) antibody disclosed herein is administered to a subject incombination with a cell expressing a chimeric antigen receptor (CAR). Incertain embodiments, the cell is a T cell. Exemplary CAR T-celltherapies are provided in Pettitt et al. (2017) Molecular Therapy26(2):342-53, which is incorporated by reference herein in its entirety.In certain embodiments, the CAR T cell therapy comprises a CD19 CAR Tcell therapy (e.g., YESCARTA™ and KYMRIAH™). In certain embodiments, thesubject has a CD19⁺ cancer (e.g., a CD19⁺ hematologic malignancy). Incertain embodiments, such CD19⁺ cancer is resistant to the CAR T celltherapy alone. In certain embodiments, subjects having a tumor orclinical indication in which the response rate to a CAR T cell therapyis no more than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less,are selected for treatment with the antibody and/or pharmaceuticalcomposition disclosed herein, alone or in combination with the CAR Tcell therapy. In certain embodiments, a subject having a diffuse largeB-cell lymphoma not otherwise specified (DLBCL-nos), primary mediastinallarge B cell lymphoma, high grade B cell lymphoma, or DLBCL arising fromfollicular lymphoma is selected for treatment with the antibody and/orpharmaceutical composition disclosed herein, alone or in combinationwith a CD19 CAR T cell therapy (e.g., YESCARTA™). In certainembodiments, a subject having B cell precursor ALL is selected fortreatment with the antibody and/or pharmaceutical composition disclosedherein, alone or in combination with a CD19 CAR T cell therapy (e.g.,KYMRIAH™). In certain embodiments, the subject has had an inadequateresponse despite exhibiting adequate expansion of infused CAR T celltherapy.

In specific embodiments, the CD19⁺ cancer is a CD19⁺ hematologicmalignancy. In certain embodiments, the CD19⁺ hematologic malignancy isa non-B-cell precursor ALL (e.g., diffuse large B-cell lymphoma ormantle cell lymphoma). In certain embodiments, the CD19⁺ cancer isresistant to or recurrent after treatment with a prior treatment (e.g.,a monoclonal anti-CD20 antibody therapy or an anthracycline containingchemotherapy regimen). In certain embodiments, the subject has aninadequate response to at least one infusion of a CD19 CAR T celltherapy (e.g., YESCARTA™ or KYMRIAH™). In certain embodiments, thesubject has a measurable disease.

In certain embodiments, an anti-CD73 (e.g., human, mouse, or cynomolgusCD73) antibody disclosed herein is administered to a subject incombination with a TCR mimic antibody. In certain embodiments, the TCRmimic antibody is an antibody that specifically binds to a peptide-MHCcomplex. For non-limiting examples of TCR mimic antibodies, see, e.g.,U.S. Pat. No. 9,074,000 and U.S. Publication Nos. US 2009/0304679 A1 andUS 2014/0134191 A1, all of which are incorporated herein by reference intheir entireties.

In certain embodiments, an anti-CD73 (e.g., human, mouse, or cynomolgusCD73) antibody disclosed herein is administered to a subject incombination with a bispecific T-cell engager (BiTE) (e.g., as describedin WO2005061547A2, which is incorporated by reference herein in itsentirety) and/or a dual-affinity re-targeting antibody (DART) (e.g., asdescribed in WO2012162067A2, which is incorporated by reference hereinin its entirety). In certain embodiments, the BiTE and/or DARTspecifically binds to a tumor-associated antigen (e.g., a polypeptideoverexpressed in a tumor, a polypeptide derived from an oncovirus, apolypeptide comprising a post-translational modification specific to atumor, a polypeptide specifically mutated in a tumor) and a molecule onan effector cell (e.g., CD3 or CD16). In certain embodiments, thetumor-associated antigen is EGFR (e.g., human EGFR), optionally whereinthe BiTE and/or DART comprises the VH and VL sequences of cetuximab. Incertain embodiments, the tumor-associated antigen is Her2 (e.g., humanHer2), optionally wherein the BiTE and/or DART comprises the VH and VLsequences of trastuzumab. In certain embodiments, the tumor-associatedantigen is CD20 (e.g., human CD20).

The anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody and theadditional therapeutic agent (e.g., chemotherapeutic, radiotherapeutic,checkpoint targeting agent, IDO inhibitor, vaccine, adjuvant, a solubleTCR, a cell expressing a TCR, a cell expressing a chimeric antigenreceptor, and/or a TCR mimic antibody) can be administered separately,sequentially or concurrently as separate dosage forms. In oneembodiment, an anti-CD73 (e.g., human, mouse, or cynomolgus CD73)antibody is administered parenterally, and an IDO inhibitor isadministered orally.

An antibody or pharmaceutical composition described herein may bedelivered to a subject by a variety of routes. These include, but arenot limited to, parenteral, intranasal, intratracheal, oral,intradermal, topical, intramuscular, intraperitoneal, transdermal,intravenous, intratumoral, conjunctival, intra-arterial, andsubcutaneous routes. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent for use as a spray. In certain embodiments, theantibody or pharmaceutical composition described herein is deliveredsubcutaneously or intravenously. In certain embodiments, the antibody orpharmaceutical composition described herein is deliveredintra-arterially. In certain embodiments, the antibody or pharmaceuticalcomposition described herein is delivered intratumorally. In certainembodiments, the antibody or pharmaceutical composition described hereinis delivered into a tumor draining lymph node.

The amount of an antibody or composition which will be effective in thetreatment and/or prevention of a condition will depend on the nature ofthe disease, and can be determined by standard clinical techniques.

The precise dose to be employed in a composition will also depend on theroute of administration, and the seriousness of the infection or diseasecaused by it, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses may also vary depending upon means of administration, target site,physiological state of the patient (including age, body weight andhealth), whether the patient is human or an animal, other medicationsadministered, or whether treatment is prophylactic or therapeutic.Usually, the patient is a human, but non-human mammals, includingtransgenic mammals, can also be treated. Treatment dosages are optimallytitrated to optimize safety and efficacy.

An anti-CD73 (e.g., human. mouse, or cynomolgus CD73) antibody describedherein can also be used to assay CD73 (e.g., human, mouse, or cynomolgusCD73) protein levels in a biological sample using classicalimmunohistological methods known to those of skill in the art, includingimmunoassays, such as the enzyme linked immunosorbent assay (ELISA),immunoprecipitation, or Western blotting. Suitable antibody assay labelsare known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹²¹In), and technetium (⁹⁹Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin. Such labels can be used to labelan antibody described herein. Alternatively, a second antibody thatrecognizes an anti-CD73 (e.g., human, mouse, or cynomolgus CD73)antibody described herein can be labeled and used in combination with ananti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody to detectCD73 (e.g., human, mouse, or cynomolgus CD73) protein levels. Therefore,in one embodiment, the present invention relates to the use of anantibody of the present invention for in vitro detection of CD73 (e.g.,human, mouse, or cynomolgus CD73) protein in a biological sample. In afurther embodiment, the present invention relates to the use of ananti-CD73 antibody of the invention, for assaying and/or detecting CD73(e.g., human, mouse, or cynomolgus CD73) protein levels in a biologicalsample in vitro, optionally wherein the anti-CD73 antibody is conjugatedto a radionuclide or detectable label, and/or carries a label describedherein, and/or wherein an immunohistological method is used.

Assaying for the expression level of CD73 (e.g., human, mouse, orcynomolgus CD73) protein is intended to include qualitatively orquantitatively measuring or estimating the level of CD73 (e.g., human,mouse, or cynomolgus CD73) protein in a first biological sample eitherdirectly (e.g., by determining or estimating absolute protein level) orrelatively (e.g., by comparing to the disease associated protein levelin a second biological sample). CD73 (e.g., human, mouse, or cynomolgusCD73) polypeptide expression level in the first biological sample can bemeasured or estimated and compared to a standard CD73 (e.g., human,mouse, or cynomolgus CD73) protein level, the standard being taken, forexample, from a second biological sample obtained from an individual nothaving the disorder or being determined by averaging levels from apopulation of individuals not having the disorder. As will beappreciated in the art, once the “standard” CD73 (e.g., human, mouse, orcynomolgus CD73) polypeptide level is known, it can be used repeatedlyas a standard for comparison. Therefore, in a further embodiment, thepresent invention relates to an in vitro method for assaying and/ordetecting CD73 protein levels, for example human CD73 protein levels, ina biological sample, comprising qualitatively or quantitativelymeasuring or estimating the level of CD73 protein, for example of humanCD73 protein, in a biological sample, by an immunohistological method.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing CD73 (e.g., human, mouse, or cynomolgusCD73). Methods for obtaining tissue biopsies and body fluids fromanimals (e.g., humans or cynomolgus monkeys) are well known in the art.Biological samples include peripheral mononuclear blood cells.

An anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody describedherein can be used for prognostic, diagnostic, monitoring and screeningapplications, including in vitro and in vivo applications well known andstandard to the skilled artisan and based on the present description.Prognostic, diagnostic, monitoring and screening assays and kits for invitro assessment and evaluation of immune system status and/or immuneresponse may be utilized to predict, diagnose and monitor to evaluatepatient samples including those known to have or suspected of having animmune system-dysfunction or with regard to an anticipated or desiredimmune system response, antigen response or vaccine response. Theassessment and evaluation of immune system status and/or immune responseis also useful in determining the suitability of a patient for aclinical trial of a drug or for the administration of a particularchemotherapeutic agent, a radiotherapeutic agent, or an antibody,including combinations thereof, versus a different agent or antibody.This type of prognostic and diagnostic monitoring and assessment isalready in practice utilizing antibodies against the HER2 protein inbreast cancer (HercepTest™, Dako) where the assay is also used toevaluate patients for antibody therapy using Herceptin®. In vivoapplications include directed cell therapy and immune system modulationand radio imaging of immune responses. Therefore, in one embodiment, thepresent invention relates to an anti-CD73 antibody and/or pharmaceuticalcomposition of the present invention for use as a diagnostic. In oneembodiment, the present invention relates to an anti-CD73 antibodyand/or pharmaceutical composition of the present invention for use in amethod for the prediction, diagnosis and/or monitoring of a subjecthaving or suspected to have an immune system-dysfunction and/or withregard to an anticipated or desired immune system response, antigenresponse or vaccine response. In another embodiment, the presentinvention relates to the use of anti-CD73 antibody of the invention, forpredicting, diagnosing and/or monitoring of a subject having orsuspected to have an immune system-dysfunction and/or with regard to ananticipated or desired immune system response, antigen response orvaccine response by assaying and/or detecting human CD73 protein levelsin a biological sample of the subject in vitro.

In one embodiment, an anti-CD73 (e.g., human, mouse, or cynomolgus CD73)antibody can be used in immunohistochemistry of biopsy samples. In oneembodiment, the method is an in vitro method. In another embodiment, ananti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody can be usedto detect levels of CD73 (e.g., human, mouse, or cynomolgus CD73), orlevels of cells which contain CD73 (e.g., human, mouse, or cynomolgusCD73) on their membrane surface, the levels of which can then be linkedto certain disease symptoms. Anti-CD73 (e.g., human, mouse, orcynomolgus CD73) antibodies described herein may carry a detectable orfunctional label and/or may be conjugated to a radionuclide ordetectable label. When fluorescence labels are used, currently availablemicroscopy and fluorescence-activated cell sorter analysis (FACS) orcombination of both methods procedures known in the art may be utilizedto identify and to quantitate the specific binding members. Anti-CD73(e.g., human, mouse, or cynomolgus CD73) antibodies described herein maycarry or may be conjugated to a fluorescence label. Exemplaryfluorescence labels include, for example, reactive and conjugatedprobes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluordyes, Cy dyes and DyLight dyes. An anti-CD73 (e.g., human, mouse, orcynomolgus CD73) antibody may carry or may be conjugated to aradioactive label or radionuclide, such as the isotopes ³H, ¹⁴C, ³²P,³⁵S, ³⁶Cl, ⁵¹Cr ⁵⁷Co, ⁵⁸C, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y ⁹⁹Tc, ¹¹¹In, ¹¹⁷Lu, ¹²¹I,¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Ac and ¹⁸⁶Re. When radioactivelabels are used, currently available counting procedures known in theart may be utilized to identify and quantitate the specific binding ofanti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody to CD73(e.g., human, mouse, or cynomolgus CD73). In the instance where thelabel is an enzyme, detection may be accomplished by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques as knownin the art. This can be achieved by contacting a sample or a controlsample with an anti-CD73 (e.g., human, mouse, or cynomolgus CD73)antibody under conditions that allow for the formation of a complexbetween the antibody and CD73 (e.g., human, mouse, or cynomolgus CD73).Any complexes formed between the antibody and CD73 (e.g., human, mouse,or cynomolgus CD73) are detected and compared in the sample and thecontrol. In light of the specific binding of the antibodies describedherein for CD73 (e.g., human, mouse, or cynomolgus CD73), the antibodiescan be used to specifically detect CD73 (e.g., human, mouse, orcynomolgus CD73) expression on the surface of cells. The antibodiesdescribed herein can also be used to purify CD73 (e.g., human, mouse, orcynomolgus CD73) via immunoaffinity purification. Also included hereinis an assay system which may be prepared in the form of a test kit, kit,or kit-of-parts for the quantitative analysis of the extent of thepresence of, for instance, CD73 (e.g., human, mouse, or cynomolgus CD73)or CD73 (e.g., human, mouse, or cynomolgus CD73)/CD73 (e.g., human,mouse, or cynomolgus CD73) ligand complexes. The system, test kit, kitor kit-of-parts may comprise a labeled component, e.g., a labeledantibody, and one or more additional immunochemical reagents.

In certain embodiments, the antibodies and pharmaceutical compositionsdescribed herein may be used or combined with one or more of achemotherapeutic agent, an anti-cancer agent, an anti-angiogenic agent,an anti-fibrotic agent, an immunotherapeutic agent, a therapeuticantibody, a bispecific antibody and “antibody-like” therapeutic protein(such as DARTs®, Duobodies®, Bites®, XmAbs®, TandAbs®, Fab derivatives),an antibody-drug conjugate (ADC), a radiotherapeutic agent, ananti-neoplastic agent, an anti-proliferation agent, an oncolytic virus,a gene modifier or editor (such as CRISPR/Cas9, zinc finger nucleases orsynthetic nucleases, TALENs), a CAR (chimeric antigen receptor) T-cellimmunotherapeutic agent, an engineered T cell receptor (TCR-T), or anycombination thereof. These therapeutic agents may be in the forms ofcompounds, antibodies, polypeptides, or polynucleotides. In certainembodiments, the application provides a product comprising a compounddescribed herein and an additional therapeutic agent as a combinedpreparation for simultaneous, separate, or sequential use in therapy,e.g. a method of treating a disease, disorder, or condition that isresponsive to CD73 inhibition and/or TGFβ signaling inhibition.

In certain embodiments, the antibodies and pharmaceutical compositionsdescribed herein may be used or combined with a DDR2 inhibitor (e.g.,dasatinib), optionally in combination with an immune checkpointinhibitor (e.g., a PD-1 inhibitor).

Targets

In certain embodiments, the antibodies and pharmaceutical compositionsdescribed herein may be used or combined with one or more of theadditional therapeutic agents. The one or more therapeutic agentsinclude, but are not limited to, an inhibitor, agonist, antagonist,ligand, modulator, stimulator, blocker, activator or suppressor of agene, ligand, receptor, protein, factor such as: Abelson murine leukemiaviral oncogene homolog 1 gene (ABL, such as ABL1), Acetyl-CoAcarboxylase (such as ACC1/2), activated CDC kinase (ACK, such as ACK1),Adenosine deaminase, adenosine receptor (such as A2B, A2a, A3),Adenylate cyclase, ADP ribosyl cyclase-1, adrenocorticotropic hormonereceptor (ACTH), Aerolysin, AKT1 gene, Alk-5 protein kinase, Alkalinephosphatase, Alpha 1 adrenoceptor, Alpha 2 adrenoceptor,Alpha-ketoglutarate dehydrogenase (KGDH), Aminopeptidase N, AMPactivated protein kinase, anaplastic lymphoma kinase (ALK, such asALK1), Androgen receptor, Angiopoietin (such as ligand-1, ligand-2),Angiotensinogen (AGT) gene, murine thymoma viral oncogene homolog 1(AKT) protein kinase (such as AKT1, AKT2, AKT3), apolipoprotein A-I(APOA1) gene, Apoptosis inducing factor, apoptosis protein (such as 1,2), apoptosis signal-regulating kinase (ASK, such as ASK1), Arginase(I), Arginine deiminase, Aromatase, Asteroid homolog 1 (ASTE1) gene,ataxia telangiectasia and Rad 3 related (ATR) serine/threonine proteinkinase, Aurora protein kinase (such as 1, 2), Axl tyrosine kinasereceptor, Baculoviral IAP repeat containing 5 (BIRC5) gene, Basigin,B-cell lymphoma 2 (BCL2) gene, Bcl2 binding component 3, Bcl2 protein,BCL2L11 gene, BCR (breakpoint cluster region) protein and gene, Betaadrenoceptor, Beta-catenin, B-lymphocyte antigen CD19, B-lymphocyteantigen CD20, B-lymphocyte cell adhesion molecule, B-lymphocytestimulator ligand, Bone morphogenetic protein-10 ligand, Bonemorphogenetic protein-9 ligand modulator, Brachyury protein, Bradykininreceptor, B-Raf proto-oncogene (BRAF), Brc-Abl tyrosine kinase,Bromodomain and external domain (BET) bromodomain containing protein(such as BRD2, BRD3, BRD4), Bruton's tyrosine kinase (BTK), Calmodulin,calmodulin-dependent protein kinase (CaMK, such as CAMKII), Cancertestis antigen 2, Cancer testis antigen NY-ESO-1, cancer/testis antigen1B (CTAG1) gene, Cannabinoid receptor (such as CB1, CB2), Carbonicanhydrase, casein kinase (CK, such as CKI, CKII), Caspase (such ascaspase-3, caspase-7, Caspase-9), caspase 8 apoptosis-related cysteinepeptidase CASP8-FADD-like regulator, Caspase recruitment domainprotein-15, Cathepsin G, CCR5 gene, CDK-activating kinase (CAK),Checkpoint kinase (such as CHK1, CHK2), chemokine (C—C motif) receptor(such as CCR2, CCR4, CCR5, CCR8), chemokine (C—X—C motif) receptor (suchas CXCR4, CXCR1 and CXCR2), Chemokine CC21 ligand, Cholecystokinin CCK2receptor, Chorionic gonadotropin, c-Kit (tyrosine-protein kinase Kit orCD117), Claudin (such as 6, 18), cluster of differentiation (CD) such asCD4, CD27, CD29, CD30, CD33, CD37, CD40, CD40 ligand receptor, CD40ligand, CD40LG gene, CD44, CD45, CD47, CD49b, CD51, CD52, CD55, CD58,CD66e, CD70 gene, CD74, CD79, CD79b, CD79B gene, CD80, CD95, CD99,CD117, CD122, CDw123, CD134, CDw137, CD158a, CD158b1, CD158b2, CD223,CD276 antigen; clusterin (CLU) gene, Clusterin, c-Met (hepatocyte growthfactor receptor (HGFR)), Complement C3, Connective tissue growth factor,COP9 signalosome subunit 5, CSF-1 (colony-stimulating factor 1receptor), CSF2 gene, CTLA-4 (cytotoxic T-lymphocyte protein 4)receptor, Cyclin D1, Cyclin G1, cyclin-dependent kinases (CDK, such asCDK1, CDK1B, CDK2-9), cyclooxygenase (such as 1, 2), CYP2B1 gene,Cysteine palmitoyltransferase porcupine, Cytochrome P450 11B2,Cytochrome P450 17, cytochrome P450 17A1, Cytochrome P450 2D6,cytochrome P450 3A4, Cytochrome P450 reductase, cytokine signalling-1,cytokine signalling-3, Cytoplasmic isocitrate dehydrogenase, Cytosinedeaminase, cytosine DNA methyltransferase, cytotoxic T-lymphocyteprotein-4, DDR2 gene, Delta-like protein ligand (such as 3, 4),Deoxyribonuclease, Dickkopf-1 ligand, dihydrofolate reductase (DHFR),Dihydropyrimidine dehydrogenase, Dipeptidyl peptidase IV, discoidindomain receptor (DDR, such as DDR1), DNA binding protein (such asHU-beta), DNA dependent protein kinase, DNA gyrase, DNAmethyltransferase, DNA polymerase (such as alpha), DNA primase, dUTPpyrophosphatase, L-dopachrome tautomerase, echinoderm microtubule likeprotein 4, EGFR tyrosine kinase receptor, Elastase, Elongation factor 1alpha 2, Elongation factor 2, Endoglin, Endonuclease, Endoplasmin,Endosialin, Endostatin, endothelin (such as ET-A, ET-B), Enhancer ofzeste homolog 2 (EZH2), Ephrin (EPH) tyrosine kinase (such as Epha3,Ephb4), Ephrin B2 ligand, epidermal growth factor, epidermal growthfactor receptors (EGFR), epidermal growth factor receptor (EGFR) gene,Epigen, Epithelial cell adhesion molecule (EpCAM), Erb-b2 (v-erb-b2avian erythroblastic leukemia viral oncogene homolog 2) tyrosine kinasereceptor, Erb-b3 tyrosine kinase receptor, Erb-b4 tyrosine kinasereceptor, E-selectin, Estradiol 17 beta dehydrogenase, Estrogen receptor(such as alpha, beta), Estrogen related receptor, Eukaryotic translationinitiation factor 5A (EIF5A) gene, Exportin 1, Extracellular signalrelated kinase (such as 1, 2), Extracellular signal-regulated kinases(ERK), Factor (such as Xa, VIIa), farnesoid x receptor (FXR), Fasligand, Fatty acid synthase (FASN), Ferritin, FGF-2 ligand, FGF-5ligand, fibroblast growth factor (FGF, such as FGF1, FGF2, FGF4),Fibronectin, Fms-related tyrosine kinase 3 (Flt3), Fms-like tyrosinekinase-3 ligand (FLT3L), focal adhesion kinase (FAK, such as FAK2),folate hydrolase prostate-specific membrane antigen 1 (FOLH1), Folatereceptor (such as alpha), Folate, Folate transporter 1, FYN tyrosinekinase, paired basic amino acid cleaving enzyme (FURIN),Beta-glucuronidase, Galactosyltransferase, Galectin-3, Ganglioside GD2,Glucocorticoid, glucocorticoid-induced TNFR-related protein GITRreceptor, Glutamate carboxypeptidase II, glutaminase, GlutathioneS-transferase P, glycogen synthase kinase (GSK, such as 3-beta),Glypican 3 (GPC3), gonadotropin-releaseing hormone (GNRH), Granulocytemacrophage colony stimulating factor (GM-CSF) receptor,Granulocyte-colony stimulating factor (GCSF) ligand, growth factorreceptor-bound protein 2 (GRB2), Grp78 (78 kDa glucose-regulatedprotein) calcium binding protein, molecular chaperone groEL2 gene, Hemeoxygenase 1 (HO1), Heat shock protein (such as 27, 70, 90 alpha, beta),Heat shock protein gene, Heat stable enterotoxin receptor, Hedgehogprotein, Heparanase, Hepatocyte growth factor, HERV-H LTR associatingprotein 2, Hexose kinase, Histamine H2 receptor, Histonemethyltransferase (DOTIL), histone deacetylase (HDAC, such as 1, 2, 3,6, 10, 11), Histone H1, Histone H3, HLA class I antigen (A-2 alpha), HLAclass II antigen, Homeobox protein NANOG, HSPB1 gene, Human leukocyteantigen (HLA), Human papillomavirus (such as E6, E7) protein, Hyaluronicacid, Hyaluronidase, Hypoxia inducible factor-1 alpha (HIF1α), ImprintedMaternally Expressed Transcript (H19) gene, mitogen-activated proteinkinase kinase kinase kinase 1 (MAP4K1), tyrosine-protein kinase HCK,I-Kappa-B kinase (IKK, such as IKKbe), IL-1 alpha, IL-1 beta, IL-12,IL-12 gene, IL-15, IL-17, IL-2 gene, IL-2 receptor alpha subunit, IL-2,IL-3 receptor, IL-4, IL-6, IL-7, IL-8, immunoglobulin (such as G, G1,G2, K, M), Immunoglobulin Fc receptor, Immunoglobulin gamma Fc receptor(such as I, III, IIIA), indoleamine 2,3-dioxygenase (IDO, such as IDO1),indoleamine pyrrole 2,3-dioxygenase 1 inhibitor, insulin receptor,Insulin-like growth factor (such as 1, 2), Integrin alpha-4/beta-1,integrin alpha-4/beta-7, Integrin alpha-5/beta-1, Integrinalpha-V/beta-3, Integrin alpha-V/beta-5, Integrin alpha-V/beta-6,Intercellular adhesion molecule 1 (ICAM-1), interferon (such as alpha,alpha 2, beta, gamma), Interferon inducible protein absent in melanoma 2(AIM2), interferon type I receptor, Interleukin 1 ligand, Interleukin 13receptor alpha 2, interleukin 2 ligand, interleukin-1receptor-associated kinase 4 (IRAK4), Interleukin-2, Interleukin-29ligand, isocitrate dehydrogenase (such as IDH1, IDH2), Janus kinase(JAK, such as JAK1, JAK2), Jun N terminal kinase, kallikrein-relatedpeptidase 3 (KLK3) gene, Killer cell Ig like receptor, Kinase insertdomain receptor (KDR), Kinesin-like protein KIF11, Kirsten rat sarcomaviral oncogene homolog (KRAS) gene, Kisspeptin (KiSS-1) receptor, KITgene, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog(KIT) tyrosine kinase, lactoferrin, Lanosterol-14 demethylase, LDLreceptor related protein-1, Leukotriene A4 hydrolase, Listeriolysin,L-Selectin, Luteinizing hormone receptor, Lyase, lymphocyte activationgene 3 protein (LAG-3), Lymphocyte antigen 75, Lymphocyte functionantigen-3 receptor, lymphocyte-specific protein tyrosine kinase (LCK),Lymphotactin, Lyn (Lck/Yes novel) tyrosine kinase, lysine demethylases(such as KDM1, KDM2, KDM4, KDM5, KDM6, A/B/C/D), Lysophosphatidate-1receptor, lysosomal-associated membrane protein family (LAMP) gene,Lysyl oxidase homolog 2, lysyl oxidase protein (LOX), lysyl oxidase-likeprotein (LOXL, such as LOXL2), Hematopoietic Progenitor Kinase 1 (HPK1),Hepatocyte growth factor receptor (MET) gene, macrophagecolony-stimulating factor (MCSF) ligand, Macrophage migration inhibitoryfact, MAGEC1 gene, MAGEC2 gene, Major vault protein, MAPK-activatedprotein kinase (such as MK2), Mas-related G-protein coupled receptor,matrix metalloprotease (MMP, such as MMP2, MMP9), Mcl-1 differentiationprotein, Mdm2 p53-binding protein, Mdm4 protein, Melan-A (MART-1)melanoma antigen, Melanocyte protein Pmel 17, melanocyte stimulatinghormone ligand, melanoma antigen family A3 (MAGEA3) gene, Melanomaassociated antigen (such as 1, 2, 3, 6), Membrane copper amine oxidase,Mesothelin, MET tyrosine kinase, Metabotropic glutamate receptor 1,Metalloreductase STEAP1 (six transmembrane epithelial antigen of theprostate 1), Metastin, methionine aminopeptidase-2, Methyltransferase,Mitochondrial 3 ketoacyl CoA thiolase, mitogen-activate protein kinase(MAPK), mitogen-activated protein kinase (MEK, such as MEK1, MEK2), mTOR(mechanistic target of rapamycin (serine/threonine kinase), mTOR complex(such as 1, 2), mucin (such as 1, 5A, 16), mut T homolog (MTH, such asMTH1), Myc proto-oncogene protein, myeloid cell leukemia 1 (MCL1) gene,myristoylated alanine-rich protein kinase C substrate (MARCKS) protein,NAD ADP ribosyltransferase, natriuretic peptide receptor C, Neural celladhesion molecule 1, Neurokinin I (NK1) receptor, Neurokinin receptor,Neuropilin 2, NF kappa B activating protein, NIMA-related kinase 9(NEK9), Nitric oxide synthase, NK cell receptor, NK3 receptor, NKG2 A Bactivating NK receptor, Noradrenaline transporter, Notch (such asNotch-2 receptor, Notch-3 receptor, Notch-4 receptor), Nuclear erythroid2-related factor 2, Nuclear Factor (NF) kappa B, Nucleolin,Nucleophosmin, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), 2oxoglutarate dehydrogenase, 2,5-oligoadenylate synthetase,O-methylguanine DNA methyltransferase, Opioid receptor (such as delta),Ornithine decarboxylase, Orotate phosphoribosyltransferase, orphannuclear hormone receptor NR4A1, Osteocalcin, Osteoclast differentiationfactor, Osteopontin, OX-40 (tumor necrosis factor receptor superfamilymember 4 TNFRSF4, or CD134) receptor, P3 protein, p38 kinase, p38 MAPkinase, p53 tumor suppressor protein, Parathyroid hormone ligand,peroxisome proliferator-activated receptors (PPAR, such as alpha, delta,gamma), P-Glycoprotein (such as 1), phosphatase and tensin homolog(PTEN), phosphatidylinositol 3-kinase (PI3K), phosphoinositide-3 kinase(PI3K such as alpha, delta, gamma), phosphorylase kinase (PK), PKN3gene, placenta growth factor, platelet-derived growth factor (PDGF, suchas alpha, beta), Platelet-derived growth factor (PDGF, such as alpha,beta), Pleiotropic drug resistance transporter, Plexin B1, PLK1 gene,polo-like kinase (PLK), Polo-like kinase 1, Poly ADP ribose polymerase(PARP, such as PARP1, 2 and 3), Preferentially expressed antigen inmelanoma (PRAME) gene, Prenyl-binding protein (PrPB), Probabletranscription factor PML, Progesterone receptor, Programmed cell death 1(PD-1), Programmed cell death ligand 1 inhibitor (PD-L1), Prosaposin(PSAP) gene, Prostanoid receptor (EP4), prostate specific antigen,Prostatic acid phosphatase, proteasome, Protein E7, Proteinfarnesyltransferase, protein kinase (PK, such as A, B, C), proteintyrosine kinase, Protein tyrosine phosphatase beta, Proto-oncogeneserine/threonine-protein kinase (PIM, such as PIM-1, PIM-2, PIM-3),P-Selectin, Purine nucleoside phosphorylase, purinergic receptor P2Xligand gated ion channel 7 (P2X₇), Pyruvate dehydrogenase (PDH),Pyruvate dehydrogenase kinase, Pyruvate kinase (PYK), 5-Alpha-reductase,Raf protein kinase (such as 1, B), RAF1 gene, Ras gene, Ras GTPase, RETgene, Ret tyrosine kinase receptor, retinoblastoma associated protein,retinoic acid receptor (such as gamma), Retinoid X receptor, Rheb (Rashomolog enriched in brain) GTPase, Rho (Ras homolog) associated proteinkinase 2, ribonuclease, Ribonucleotide reductase (such as M2 subunit),Ribosomal protein S6 kinase, RNA polymerase (such as I, II), Ron(Recepteur d'Origine Nantais) tyrosine kinase, ROS1 (ROS proto-oncogene1, receptor tyrosine kinase) gene, Ros1 tyrosine kinase, Runt-relatedtranscription factor 3, Gamma-secretase, S100 calcium binding proteinA9, Sarco endoplasmic calcium ATPase, Second mitochondria-derivedactivator of caspases (SMAC) protein, Secreted frizzled relatedprotein-2, Semaphorin-4D, Serine protease, serine/threonine kinase(STK), serine/threonine-protein kinase (TBK, such as TBK1), signaltransduction and transcription (STAT, such as STAT-1, STAT-3, STAT-5),Signaling lymphocytic activation molecule (SLAM) family member 7,six-transmembrane epithelial antigen of the prostate (STEAP) gene, SLcytokine ligand, smoothened (SMO) receptor, Sodium iodide cotransporter,Sodium phosphate cotransporter 2B, Somatostatin receptor (such as 1, 2,3, 4, 5), Sonic hedgehog protein, Son of sevenless (SOS), Specificprotein 1 (Sp1) transcription factor, Sphingomyelin synthase,Sphingosine kinase (such as 1, 2), Sphingosine-1-phosphate receptor-1,spleen tyrosine kinase (SYK), SRC gene, Src tyrosine kinase, STAT3 gene,Steroid sulfatase, Stimulator of interferon genes (STING) receptor,stimulator of interferon genes protein, Stromal cell-derived factor 1ligand, SUMO (small ubiquitin-like modifier), Superoxide dismutase,Survivin protein, Synapsin 3, Syndecan-1, Synuclein alpha, T cellsurface glycoprotein CD28, tank-binding kinase (TBK), TATA box-bindingprotein-associated factor RNA polymerase I subunit B (TAF1B) gene,T-cell CD3 glycoprotein zeta chain, T-cell differentiation antigen CD6,T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), T-cellsurface glycoprotein CD8, Tec protein tyrosine kinase, Tek tyrosinekinase receptor, telomerase, Telomerase reverse transcriptase (TERT)gene, Tenascin, TGF beta 2 ligand, Thrombopoietin receptor, Thymidinekinase, Thymidine phosphorylase, Thymidylate synthase, Thymosin (such asalpha 1), Thyroid hormone receptor, Thyroid stimulating hormonereceptor, Tissue factor, TNF related apoptosis inducing ligand, TNFR1associated death domain protein, TNF-related apoptosis-inducing ligand(TRAIL) receptor, TNFSF11 gene, TNFSF9 gene, Toll-like receptor (TLRsuch as 1-13), topoisomerase (such as I, II, III), Transcription factor,Transferase, Transferrin, Transforming growth factor (TGF, such as beta)kinase, Transforming growth factor TGF-β receptor kinase,Transglutaminase, Translocation associated protein, Transmembraneglycoprotein NMB, Trop-2 calcium signal transducer, trophoblastglycoprotein (TPBG) gene, Trophoblast glycoprotein, Tropomyosin receptorkinase (Trk) receptor (such as TrkA, TrkB, TrkC), Tryptophan5-hydroxylase, Tubulin, Tumor necrosis factor (TNF, such as alpha,beta), Tumor necrosis factor 13C receptor, tumor progression locus 2(TPL2), Tumor protein 53 (TP53) gene, Tumor suppressor candidate 2(TUSC2) gene, Tumor specific neoantigens, Tyrosinase, Tyrosinehydroxylase, tyrosine kinase (TK), Tyrosine kinase receptor, Tyrosinekinase with immunoglobulin-like and EGF-like domains (TIE) receptor,Tyrosine protein kinase ABL1 inhibitor, Ubiquitin, Ubiquitin carboxylhydrolase isozyme L5, Ubiquitin thioesterase-14, Ubiquitin-conjugatingenzyme E21 (UBE2I, UBC9), Urease, Urokinase plasminogen activator,Uteroglobin, Vanilloid VR1, Vascular cell adhesion protein 1, vascularendothelial growth factor receptor (VEGFR), V-domain Ig suppressor ofT-cell activation (VISTA), VEGF-1 receptor, VEGF-2 receptor, VEGF-3receptor, VEGF-A, VEGF-B, Vimentin, Vitamin D3 receptor, Proto-oncogenetyrosine-protein kinase Mer, Yes, Wee-1 protein kinase, Wilms' tumorantigen 1, Wilms' tumor protein, X-linked inhibitor of apoptosisprotein, Zinc finger protein transcription factor or any combinationthereof.

Mechanism of Action

The antibodies and pharmaceutical compositions described herein may beused or combined with one or more of the additional therapeutic agents.Therapeutic agents may be categorized by their mechanism of action into,for example, the following groups:

-   -   anti-metabolites/anti-cancer agents, such as pyrimidine analogs        floxuridine, capecitabine, cytarabine, CPX-351 (liposomal        cytarabine, daunorubicin), and TAS-118;    -   purine analogs, folate antagonists (such as pralatrexate), and        related inhibitors;    -   antiproliferative/antimitotic agents including natural products,        such as vinca alkaloids (vinblastine, vincristine) and        microtubule disruptors such as taxane (paclitaxel, docetaxel),        vinblastin, nocodazole, epothilones, vinorelbine (NAVELBINE®),        and epipodophyllotoxins (etoposide, teniposide);    -   DNA damaging agents, such as actinomycin, amsacrine, busulfan,        carboplatin, chlorambucil, cisplatin, cyclophosphamide        (CYTOXAN®), dactinomycin, daunorubicin, doxorubicin, epirubicin,        iphosphamide, melphalan, merchlorethamine, mitomycin C,        mitoxantrone, nitrosourea, procarbazine, taxol, Taxotere,        teniposide, etoposide, and triethylenethiophosphoramide;    -   DNA-hypomethylating agents, such as guadecitabine (SGI-110),        ASTX727;    -   antibiotics such as dactinomycin, daunorubicin, doxorubicin,        idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin        (mithramycin);    -   enzymes such as L-asparaginase which systemically metabolizes        L-asparagine and deprives cells which do not have the capacity        to synthesize their own asparagine;    -   antiplatelet agents;    -   DNAi oligonucleotides targeting Bcl-2, such as PNT2258;    -   agents that activate or reactivate latent human immunodeficiency        virus (HIV), such as panobinostat and romidepsin;    -   asparaginase stimulators, such as crisantaspase (Erwinase®) and        GRASPA (ERY-001, ERY-ASP), calaspargase pegol;    -   pan-Trk, ROS1 and ALK inhibitors, such as entrectinib, TPX-0005;    -   anaplastic lymphoma kinase (ALK) inhibitors, such as alectinib,        ceritinib;    -   antiproliferative/antimitotic alkylating agents, such as        nitrogen mustard cyclophosphamide and analogs (melphalan,        chlorambucil, hexamethylmelamine, thiotepa), alkyl nitrosoureas        (carmustine) and analogs, streptozocin, and triazenes        (dacarbazine);    -   antiproliferative/antimitotic antimetabolites, such as folic        acid analogs (methotrexate);    -   platinum coordination complexes (cisplatin, oxiloplatinim, and        carboplatin), procarbazine, hydroxyurea, mitotane, and        aminoglutethimide;    -   hormones, hormone analogs (estrogen, tamoxifen, goserelin,        bicalutamide, and nilutamide), and aromatase inhibitors        (letrozole and anastrozole);    -   anticoagulants such as heparin, synthetic heparin salts, and        other inhibitors of thrombin;    -   fibrinolytic agents such as tissue plasminogen activator,        streptokinase, urokinase, aspirin, dipyridamole, ticlopidine,        and clopidogrel;    -   antimigratory agents;    -   antisecretory agents (brefeldin);    -   immunosuppressives, such as tacrolimus, sirolimus, azathioprine,        and mycophenolate;    -   growth factor inhibitors, and vascular endothelial growth factor        inhibitors;    -   fibroblast growth factor inhibitors, such as FPA14;    -   anti-VEGFR antibodies, such as IMC-3C5, GNR-011, tanibirumab;    -   anti-VEGF/DDL4 antibodies, such as ABT-165;    -   anti-cadherins antibodies, such as HKT-288;    -   anti-CD70 antibodies, such as AMG-172; anti-leucine-rich repeat        containing 15 (LRRC15) antibodies, such as ABBV-085. ARGX-110;    -   angiotensin receptor blockers, nitric oxide donors;    -   antisense oligonucleotides, such as AEG35156, IONIS-KRAS-2.5Rx,        EZN-3042, RX-0201, IONIS-AR-2.5Rx, BP-100 (prexigebersen),        IONIS-STAT3-2.5Rx;    -   DNA interference oligonucleotides, such as PNT2258, AZD-9150;    -   anti-ANG-2 antibodies, such as MEDI3617, and LY3127804;    -   anti-ANG-1/ANG-2 antibodies, such as AMG-780;    -   anti-MET/EGFR antibodies, such as LY3164530;    -   anti-EGFR antibodies, such as ABT-414, AMG-595, necitumumab,        ABBV-221, depatuxizumab mafodotin (ABT-414), tomuzotuximab,        ABT-806, vectibix, modotuximab, RM-1929;    -   anti-CSF1R antibodies, such as emactuzumab, LY3022855, AMG-820,        FPA-008 (cabiralizumab);    -   anti-CD40 antibodies, such as RG7876, SEA-CD40, APX-005M,        ABBV-428;    -   anti-endoglin antibodies, such as TRC105 (carotuximab);    -   anti-CD45 antibodies, such as 131I-BC8 (lomab-B);    -   anti-HER3 antibodies, such as LJM716, GSK2849330;    -   anti-HER2 antibodies, such as margetuximab, MEDI4276, BAT-8001;    -   anti-HLA-DR antibodies, such as IMMU-114;    -   anti-TL-3 antibodies, such as JNJ-56022473;    -   anti-OX40 antibodies, such as MEDI6469, MEDI6383, MEDI0562        (tavolixizumab), MOXR0916, PF-04518600, RG-7888, GSK-3174998,        BMS-986178, GBR-8383, ABBV-368;    -   anti-EphA3 antibodies, such as KB-004;    -   anti-CD20 antibodies, such as obinutuzumab, IGN-002;    -   anti-CD20/CD3 antibodies, such as RG7828;    -   anti-CD37 antibodies, such as AGS67E, otlertuzumab (TRU-016);    -   anti-ENPP3 antibodies, such as AGS-16C3F;    -   anti-FGFR-3 antibodies, such as LY3076226, B-701;    -   anti-FGFR-2 antibodies, such as GAL-F2;    -   anti-C5 antibodies, such as ALXN-1210;    -   anti-CD27 antibodies, such as varlilumab (CDX-1127);    -   anti-TROP-2 antibodies, such as IMMU-132    -   anti-NKG2a antibodies, such as monalizumab;    -   anti-VISTA antibodies, such as HMBD-002;    -   anti-PVRIG antibodies, such as COM-701;    -   anti-EpCAM antibodies, such as VB4-845;    -   anti-BCMA antibodies, such as GSK-2857916    -   anti-CEA antibodies, such as RG-7813;    -   anti-cluster of differentiation 3 (CD3) antibodies, such as        MGD015;    -   anti-folate receptor alpha antibodies, such as IMGN853;    -   epha2 inhibitors, such as MM-310;    -   anti LAG-3 antibodies, such as relatlimab (ONO-4482), LAG-525,        MK-4280, REGN-3767;    -   raf kinase/VEGFR inhibitors, such as RAF-265;    -   polycomb protein (EED) inhibitors, such as MAK683;    -   anti-fibroblast activation protein (FAP)/IL-2R antibodies, such        as RG7461;    -   anti-fibroblast activation protein (FAP)/TRAIL-R2 antibodies,        such as RG7386;    -   anti-fucosyl-GM1 antibodies, such as BMS-986012;    -   p38 MAP kinase inhibitors, such as ralimetinib;    -   PRMT1 inhibitors, such as MS203;    -   Sphingosine kinase 2 (SK2) inhibitors, such as opaganib;    -   FLT3-ITD inhibitors, such as BCI-332;    -   FLT3-ITD/Mer tyrosine kinase inhibitors, such as MRX-2843;    -   Nuclear erythroid 2-related factor 2 stimulators, such as        omaveloxolone (RTA-408);    -   Tropomyosin receptor kinase (TRK) inhibitors, such as LOXO-195,        ONO-7579;    -   anti-ICOS antibodies, such as JTX-2011, GSK3359609;    -   anti-DR5 (TRAIL2) antibodies, such as DS-8273;    -   anti-GD2 antibodies, such as APN-301;    -   anti-interleukin-17 (IL-17) antibodies, such as CJM-112;    -   anti-carbonic anhydrase IX antibodies, such as TX-250;    -   anti-CD38-attenukine, such as TAK573;    -   anti-Mucin 1 antibodies, such as gatipotuzumab;    -   anti-FTL3 antibodies, such as Flysyn, ASP-1235;    -   anti-FLT3/CD3 BiTE antibodies, such as AMG-427;    -   Mucin 1 inhibitors, such as GO-203-2C;    -   MARCKS protein inhibitors, such as BIO-11006;    -   Folate antagonists, such as arfolitixorin;    -   Galectin-3 inhibitors, such as GR-MD-02;    -   Phosphorylated P68 inhibitors, such as RX-5902;    -   CD95/TNF modulators, such as ofranergene obadenovec;    -   PI3K/Akt/mTOR inhibitors, such as ABTL-0812;    -   pan-PIM kinase inhibitors, such as INCB-053914;    -   PIM/FLT3 kinase inhibitors, such as MEN-1703 (SEL-24);    -   IL-12 gene stimulators, such as EGEN-001, tavokinogene        telseplasmid;    -   Heat shock protein HSP90 inhibitors, such as TAS-116, PEN-866;    -   VEGF/HGF antagonists, such as MP-0250;    -   SYK tyrosine kinase/FLT3 tyrosine kinase inhibitors, such as        TAK-659 (mivavotinib);    -   SYK tyrosine kinase/JAK tyrosine kinase inhibitors, such as        ASN-002;    -   JAK3/JAK1/TBK1 kinase inhibitors, such as CS-12912;    -   FLT3 tyrosine kinases, such as FF-10101, HM-43239, SKI-G-801;    -   FMS-like tyrosine kinase-3 ligand (FLT3L), such as CDX-301;    -   EGFR/FLT3/ABL tyrosine kinase inhibitors, such as SKLB-1028;    -   FLT3/MEK1 inhibitors, such as E-6201;    -   IL-24 antagonist, such as AD-IL24;    -   RIG-I agonists, such as RGT-100;    -   Aerolysin stimulators, such as topsalysin;    -   P-Glycoprotein 1 inhibitors, such as HM-30181A;    -   CSF-1 antagonists, such as ARRY-382, BLZ-945;    -   CCR8 inhibitors, such as I-309, SB-649701, HG-1013, RAP-310;    -   anti-Mesothelin antibodies, such as SEL-403;    -   Thymidine kinase stimulators, such as aglatimagene besadenovec;    -   Polo-like kinase 1 inhibitors, such as PCM-075;    -   TLR-7 agonists, such as TMX-101 (imiquimod);    -   NEDD8 inhibitors, such as pevonedistat (MLN-4924), TAS-4464;        Pleiotropic pathway modulators, such as avadomide (CC-122);    -   FoxM1 inhibitors, such as thiostrepton;    -   Anti-MUC1 antibodies, such as Mab-AR-20.5;    -   anti-CD38 antibodies, such as isatuximab, MOR-202;    -   UBA1 inhibitors, such as TAK-243;    -   Src tyrosine kinase inhibitors, such as VAL-201;    -   VDAC/HK inhibitors, such as VDA-1102;    -   BRAF/PI3K inhibitors, such as ASN-003;    -   Elf4a inhibitors, such as rohinitib, eFT226;    -   TP53 gene stimulators, such as ad-p53;    -   PD-L1/EGFR inhibitors, such as GNS-1480 (lazertinib);    -   PD-1/CTLA-4 inhibitors, such as PF-06936308;    -   Retinoic acid receptor alpha (RARa) inhibitors, such as SY-1425;    -   SIRT3 inhibitors, such as YC8-02;    -   Stromal cell-derived factor 1 ligand inhibitors, such as        olaptesed pegol (NOX-A12);    -   IL-4 receptor modulators, such as MDNA-55;    -   Arginase-I stimulators, such as pegzilarginase;    -   Topoisomerase I inhibitor/hypoxia inducible factor-1 alpha        inhibitors, such as PEG-SN38 (firtecan pegol);    -   Hypoxia inducible factor-1 alpha inhibitors, such as PT-2977,        PT-2385;    -   CD122 agonists such as NKTR-214;    -   p53 tumor suppressor protein stimulators such as kevetrin;    -   Mdm4/Mdm2 p53-binding protein inhibitors, such as ALRN-6924;    -   kinesin spindle protein (KSP) inhibitors, such as filanesib        (ARRY-520);    -   CD80-fc fusion protein inhibitors, such as FPT-155;    -   Menin and mixed lineage leukemia (MLL) inhibitors such as        KO-539;    -   Liver x receptor agonists, such as RGX-104;    -   IL-10 agonists, such as AM-0010;    -   EGFR/ErbB-2 inhibitors, such as varlitinib;    -   VEGFR/PDGFR inhibitors, such as vorolanib;    -   IRAK4 inhibitors, such as CA-4948;    -   anti-TLR-2 antibodies, such as OPN-305;    -   Calmodulin modulators, such as CBP-501;    -   Glucocorticoid receptor antagonists, such as relacorilant        (CORT-125134);    -   Second mitochondria-derived activator of caspases (SMAC) protein        inhibitors, such as BI-891065;    -   Lactoferrin modulators, such as LTX-315;    -   Kit tyrosine kinase/PDGF receptor alpha antagonists such as        DCC-2618;    -   KIT inhibitors, such as PLX-9486;    -   Exportin 1 inhibitors, such as eltanexor;    -   EGFR/ErbB2/Ephb4 inhibitors, such as tesevatinib;    -   anti-CD33 antibodies, such as IMGN-779;    -   anti-KMA antibodies, such as MDX-1097;    -   anti-TIM-3 antibodies, such as TSR-022, LY-3321367, MBG-453;    -   anti-CD55 antibodies, such as PAT-SC1;    -   anti-PSMA antibodies, such as ATL-101;    -   anti-CD100 antibodies, such as VX-15;    -   anti-EPHA3 antibodies, such as fibatuzumab;    -   anti-Erbb antibodies, such as CDX-3379, HLX-02, seribantumab;    -   anti-APRIL antibodies, such as BION-1301;    -   Anti-Tigit antibodies, such as BMS-986207, RG-6058;    -   CHST15 gene inhibitors, such as STNM-01;    -   RAS inhibitors, such as NEO-100;    -   Somatostatin receptor antagonist, such as OPS-201;    -   CEBPA gene stimulators, such as MTL-501;    -   DKK3 gene modulators, such as MTG-201;    -   p70s6k inhibitors, such as MSC2363318A;    -   methionine aminopeptidase 2 (MetAP2) inhibitors, such as M8891,        APL-1202;    -   arginine N-methyltransferase 5 inhibitors, such as GSK-3326595;    -   anti-programmed cell death protein 1 (anti-PD-1) antibodies,        such as nivolumab (OPDIVO®, BMS-936558, MDX-1106), pembrolizumab        (KEYTRUDA®, MK-3477, SCH-900475, lambrolizumab, CAS Reg. No.        1374853-91-4), pidilizumab, PF-06801591, BGB-A317        (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), CS-1003,        HLX-10, MGA-012, BI-754091, REGN-2810 (cemiplimab), JS-001        (toripalimab), JNJ-63723283, genolimzumab (CBT-501), LZM-009,        BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021,        ABBV-181, AK-105, PD1-PIK, BAT-1306, and anti-programmed        death-ligand 1 (anti-PD-L1) antibodies such as BMS-936559,        atezolizumab (MPDL3280A), durvalumab (MEDI-4736), avelumab,        CK-301, (MSB0010718C), MEDI-0680, CX-072, CBT-502, PDR-001        (spartalizumab), TSR-042 (dostarlimab), MSB-2311, JTX-4014,        BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308        (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001),        BCD-135, FAZ-053, and MDX1105-01;    -   PD-L1/VISTA antagonists such as CA-170;    -   PD-1/PD-L1 inhibitors, such as GS-4416, INCB086550, GS-4224;    -   anti-PD-L1/TGFβ antibodies, such as M-7824;    -   anti-transferrin antibodies, such as CX-2029;    -   anti-IL-8 (Interleukin-8) antibodies, such as HuMax-Inflam;    -   ATM (ataxia telangiectasia) inhibitors, such as AZD0156;    -   CHK1 inhibitors, such as GDC-0575, LY2606368 (prexasertib),        SRA737, RG7741 (CHK1/2);    -   CXCR4 antagonists, such as BL-8040, LY2510924, burixafor        (TG-0054), X₄P-002, X₄P-001-IO;    -   EXH2 inhibitors, such as GSK2816126; HER2 inhibitors, such as        neratinib, tucatinib (ONT-380);    -   KDM1 inhibitors, such as ORY-1001, IMG-7289, INCB-59872,        GSK-2879552;    -   CXCR2 antagonists, such as AZD-5069;    -   GM-CSF antibodies, such as lenzilumab;    -   DNA dependent protein kinase inhibitors, such as MSC2490484A        (nedisertib), VX-984, AsiDNA (DT-01);    -   protein kinase C (PKC) inhibitors, such as LXS-196,        sotrastaurin;    -   Selective estrogen receptor downregulators (SERD), such as        fulvestrant (Faslodex®), RG6046, RG6047, elacestrant (RAD-1901)        and AZD9496;    -   Selective estrogen receptor covalent antagonists (SERCAs), such        as H3B-6545;    -   selective androgen receptor modulator (SARM), such as GTX-024,        darolutamide;    -   transforming growth factor-beta (TGF-beta) kinase antagonists,        such as galunisertib;    -   anti-transforming growth factor-beta (TGF-beta) antibodies, such        as LY3022859, NIS793, XOMA 089;    -   bispecific antibodies, such as MM-141 (IGF-1/ErbB3), MM-111        (Erb2/Erb3), JNJ-64052781 (CD19/CD3), PRS-343 (CD-137/HER2),        AFM26 (BCMA/CD16A), JNJ-61186372 (EGFR/cMET), AMG-211 (CEA/CD3),        RG7802 (CEA/CD3), ERY-974 (CD3/GPC3) vancizumab        (angiopoietins/VEGF), PF-06671008 (Cadherins/CD3), AFM-13        (CD16/CD30), APV0436 (CD123/CD3), flotetuzumab (CD123/CD3),        REGN-1979 (CD20/CD3), MCLA-117 (CD3/CLEC12A), MCLA-128        (HER2/IER3), JNJ-0819, JNJ-7564 (CD3/heme), AMG-757 (DLL3-CD3),        MGD-013 (PD-1/LAG-3), FS-118 (LAG-3/PD-L1) MGD-019        (PD-1/CTLA-4), KN-046 (PD-1/CTLA-4), MEDI-5752 (CTLA-4/PD-1),        RO-7121661 (PD-1/TIM-3), XmAb-20717 (PD-1/CTLA-4), AK-104        (CTLA-4/PD-1), AMG-330 (CD33/CD3), AMG-420 (BCMA/CD3), BI-836880        (VEFG/ANG2), JNJ-63709178 (CD123/CD3), MGD-007 (CD3/gpA33), and        MGD-009 (CD3/B7H3);    -   Mutant selective EGFR inhibitors, such as PF-06747775, EGF816        (nazartinib), ASP8273, ACEA-0010, BI-1482694;    -   Anti-GITR (glucocorticoid-induced tumor necrosis factor        receptor-related protein) antibodies, such as MEDI1873, FPA-154,        TRX-518, BMS-986156, MK-1248, GWN-323;    -   anti-delta-like protein ligand 3 (DDL3) antibodies, such as        rovalpituzumab tesirine;    -   anti-clusterin antibodies, such as AB-16B5;    -   anti-Ephrin-A4 (EFNA4) antibodies, such as PF-06647263;    -   anti-RANKL antibodies, such as denosumab;    -   anti-mesothelin antibodies, such as BMS-986148, Anti-MSLN-MMAE;    -   anti-sodium phosphate cotransporter 2B (NaP2B) antibodies, such        as lifastuzumab    -   anti-c-Met antibodies, such as ABBV-399;    -   Adenosine A2A receptor antagonists, such as CPI-444, AZD-4635,        preladenant, PBF-509;    -   Alpha-ketoglutarate dehydrogenase (KGDH) inhibitors, such as        CPI-613;    -   XPO1 inhibitors, such as selinexor (KPT-330);    -   Isocitrate dehydrogenase 2 (IDH2) inhibitors, such as enasidenib        (AG-221);    -   IDH1 inhibitors such as AG-120, and AG-881 (IDH1 and IDH2),        IDH-305, BAY-1436032;    -   interleukin-3 receptor (IL-3R) modulators, such as SL-401;    -   Arginine deiminase stimulators, such as pegargiminase        (ADI-PEG-20);    -   antibody-drug conjugates, such as MLN0264 (anti-GCC, guanylyl        cyclase C), T-DM1 (trastuzumab emtansine, Kadcycla),        milatuzumab-doxorubicin (hCD74-DOX), brentuximab vedotin,        DCDT2980S, polatuzumab vedotin, SGN-CD70A, SGN-CD19A, inotuzumab        ozogamicin, lorvotuzumab mertansine, SAR3419, isactuzumab        govitecan, enfortumab vedotin (ASG-22ME), ASG-15ME, DS-8201        ((trastuzumab deruxtecan), 225Ac-lintuzumab, U3-1402,        177Lu-tetraxetan-tetuloma, tisotumab vedotin, anetumab        ravtansine, CX-2009, SAR-566658, W-0101, polatuzumab vedotin,        ABBV-085;    -   claudin-18 inhibitors, such as claudiximab;    -   β-catenin inhibitors, such as CWP-291;    -   anti-CD73 antibodies, such as MEDI-9447 (oleclumab), CPX-006,        IPH-53, BMS-986179, NZV-930;    -   CD73 inhibitors, such as AB-680, PSB-12379, PSB-12441,        PSB-12425, CB-708;    -   CD39/CD73 inhibitors, such as PBF-1662;    -   chemokine receptor 2 (CCR) inhibitors, such as PF-04136309,        CCX-872, BMS-813160 (CCR2/CCR5)    -   thymidylate synthase inhibitors, such as ONX-0801;    -   ALK/ROS1 inhibtors, such as lorlatinib;    -   tankyrase inhibitors, such as G007-LK;    -   Mdm2 p53-binding protein inhibitors, such as CMG-097, HDM-201;    -   c-PTM inhibitors, such as PIM447;    -   BRAF inhibitors, such as dabrafenib, vemurafenib, encorafenib        (LGX818), PLX8394;    -   sphingosine kinase-2 (SK2) inhibitors, such as Yeliva®        (ABC294640);    -   cell cycle inhibitors, such as selumetinib (MEK1/2), and        sapacitabine;    -   AKT inhibitors such as MK-2206, ipatasertib, afuresertib,        AZD5363, and ARQ-092, capivasertib, triciribine;    -   anti-CTLA-4 (cytotoxic T-lymphocyte protein-4) inhibitors, such        as tremelimumab, BMS-986218;    -   c-MET inhibitors, such as AMG-337, savolitinib, tivantinib        (ARQ-197), capmatinib, and tepotinib, ABT-700, AG213, AMG-208,        JNJ-38877618 (OMO-1), merestinib, HQP-8361;    -   c-Met/VEGFR inhibitors, such as BMS-817378, TAS-115;    -   c-Met/RON inhibitors, such as BMS-777607;    -   c-Met/VEGF2/AXL/RON/Mer/FLT3 inhibitors, such as CT-053        (ningetinib);    -   c-Kit/VEGFR2/PDGFR/VEGFR3/FLT1/FLT3 inhibitors, such as SHR-1020        (famitinib L-malate);    -   BRAF/EGFR inhibitors, such as BGB-283;    -   bcr/abl inhibitors, such as rebastinib, asciminib;    -   MNK1/MNIK2 inhibitors, such as eFT-508;    -   mTOR inhibitor/cytochrome P450 3A4 stimulators, such as TYME-88    -   lysine-specific demethylase-1 (LSD1) inhibitors, such as        CC-90011;    -   Pan-RAF inhibitors, such as LY3009120, LXH254, TAK-580;    -   Raf/MEK inhibitors, such as RG7304;    -   CSF1R/KIT and FLT3 inhibitors, such as pexidartinib (PLX3397);    -   kinase inhibitors, such as vandetanib;    -   E selectin antagonists, such as GMI-1271;    -   differentiation inducers, such as tretinoin;    -   epidermal growth factor receptor (EGFR) inhibitors, such as        osimertinib (AZD-9291);    -   topoisomerase inhibitors, such as doxorubicin, daunorubicin,        dactinomycin, eniposide, epirubicin, etoposide, idarubicin,        irinotecan, mitoxantrone, pixantrone, sobuzoxane, topotecan,        irinotecan, MM-398 (liposomal irinotecan), vosaroxin and        GPX-150, aldoxorubicin, AR-67, mavelertinib, AST-2818, avitinib        (ACEA-0010), irofulven (MGI-114);    -   corticosteroids, such as cortisone, dexamethasone,        hydrocortisone, methylprednisolone, prednisone, prednisolone;    -   growth factor signal transduction kinase inhibitors;    -   nucleoside analogs, such as DFP-10917;    -   Axl inhibitors, such as BGB-324 (bemcentinib), SLC-0211;    -   BET inhibitors, such as INCB-054329, INCB057643, TEN-010,        AZD-5153, ABT-767, BMS-986158, CC-90010, GSK525762 (molibresib),        NHWD-870, ODM-207, GSK-2820151, GSK-1210151A, ZBC246, ZBC260,        ZEN3694, FT-1101, RG-6146, CC-90010, mivebresib, BI-894999,        PLX-2853, PLX-51107, CPI-0610, GS-5829;    -   PARP inhibitors, such as olaparib, rucaparib, veliparib,        talazoparib, ABT-767, BGB-290;    -   Proteasome inhibitors, such as ixazomib, carfilzomib        (Kyprolis®), marizomib;    -   Glutaminase inhibitors, such as CB-839;    -   Vaccines, such as peptide vaccine TG-01 (RAS), GALE-301,        GALE-302, nelipepimut-s, SurVaxM, DSP-7888, TPIV-200, PVX-410,        VXL-100, DPX-E7, ISA-101, 6MHP, OSE-2101, galinpepimut-S,        SVN53-67/M57-KLH, INIU-131; bacterial vector vaccines such as        CRS-207/GVAX, axalimogene filolisbac (ADXS11-001); adenovirus        vector vaccines such as nadofaragene firadenovec; autologous        Gp96 vaccine; dendritic cells vaccines, such as CVactm,        stapuldencel-T, eltrapuldencel-T, SL-701, BSK01TM,        rocapuldencel-T (AGS-003), DCVAC, CVactm, stapuldencel-T,        eltrapuldencel-T, SL-701, BSK01TM, ADXS31-142; oncolytic        vaccines such as, talimogene laherparepvec, pexastimogene        devacirepvec, GL-ONC1, MG1-MA3, parvovirus H-1, ProstAtak,        enadenotucirev, MG1MA3, ASN-002 (TG-1042); therapeutic vaccines,        such as CVAC-301, CMP-001, PF-06753512, VBI-1901, TG-4010,        ProscaVax™; tumor cell vaccines, such as Vigil® (IND-14205),        Oncoquest-L vaccine; live attenuated, recombinant, serotype 1        poliovirus vaccine, such as PVS-RIPO; Adagloxad simolenin;        MEDI-0457; DPV-001 a tumor-derived, autophagosome enriched        cancer vaccine; RNA vaccines such as, CV-9209, LV-305; DNA        vaccines, such as MEDI-0457, MVI-816, INO-5401; modified        vaccinia virus Ankara vaccine expressing p53, such as MVA-p53;        DPX-Survivac; BriaVax™; GI-6301; GI-6207; GI-4000;    -   anti-DLL4 (delta like ligand 4) antibodies, such as demcizumab;    -   STAT-3 inhibitors, such as napabucasin (BBI-608);    -   ATPase p97 inhibitors, such as CB-5083;    -   smoothened (SMO) receptor inhibitors, such as Odomzo®        (sonidegib, formerly LDE-225), LEQ506, vismodegib (GDC-0449),        BMS-833923, glasdegib (PF-04449913), LY2940680, and        itraconazole;    -   interferon alpha ligand modulators, such as interferon alpha-2b,        interferon alpha-2a biosimilar (Biogenomics), ropeginterferon        alfa-2b (AOP-2014, P-1101, PEG IFN alpha-2b), Multiferon        (Alfanative, Viragen), interferon alpha 1b, Roferon-A (Canferon,        Ro-25-3036), interferon alfa-2a follow-on biologic        (Biosidus)(Inmutag, Inter 2A), interferon alfa-2b follow-on        biologic (Biosidus—Bioferon, Citopheron, Ganapar, Beijing Kawin        Technology—Kaferon), Alfaferone, pegylated interferon alpha-1b,        peginterferon alfa-2b follow-on biologic (Amega), recombinant        human interferon alpha-1b, recombinant human interferon        alpha-2a, recombinant human interferon alpha-2b, veltuzumab-IFN        alpha 2b conjugate, Dynavax (SD-101), and interferon alfa-n1        (Humoferon, SM-10500, Sumiferon);    -   interferon gamma ligand modulators, such as interferon gamma        (OH-6000, Ogamma 100);    -   IL-6 receptor modulators, such as tocilizumab, siltuximab,        AS-101 (CB-06-02, IVX-Q-101);    -   Telomerase modulators, such as, tertomotide (GV-1001, HR-2802,        Riavax) and imetelstat (GRN-163, JNJ-63935937);    -   DNA methyltransferases inhibitors, such as temozolomide        (CCRG-81045), decitabine, guadecitabine (S-110, SGI-110),        KRX-0402, RX-3117, RRx-001, and azacitidine;    -   DNA gyrase inhibitors, such as pixantrone and sobuzoxane;    -   Bcl-2 family protein inhibitors, such as ABT-263, venetoclax        (ABT-199), ABT-737, and AT-101;    -   Notch inhibitors, such as LY3039478 (crenigacestat), tarextumab        (anti-Notch2/3), BMS-906024;    -   anti-myostatin inhibitors, such as landogrozumab;    -   hyaluronidase stimulators, such as PEGPH-20;    -   Wnt pathway inhibitors, such as SM-04755, PRI-724, WNT-974;    -   gamma-secretase inhibitors, such as PF-03084014, MK-0752,        RO-4929097;    -   Grb-2 (growth factor receptor bound protein-2) inhibitors, such        as BP1001;    -   TRAIL pathway-inducing compounds, such as ONC201, ABBV-621;    -   Focal adhesion kinase inhibitors, such as VS-4718, defactinib,        GSK2256098;    -   hedgehog inhibitors, such as saridegib, sonidegib (LDE225),        glasdegib and vismodegib;    -   Aurora kinase inhibitors, such as alisertib (MLN-8237), and        AZD-2811, AMG-900, barasertib, ENMD-2076;    -   HSPB1 modulators (heat shock protein 27, HSP27), such as        brivudine, apatorsen;    -   ATR inhibitors, such as BAY-937, AZD6738, AZD6783, VX-803,        VX-970 (berzosertib) and VX-970;    -   mTOR inhibitors, such as sapanisertib and vistusertib (AZD2014),        ME-344;    -   mTOR/PI3K inhibitors, such as gedatolisib, GSK2141795,        omipalisib, RG6114;    -   Hsp90 inhibitors, such as AUY922, onalespib (AT13387), SNX-2112,        SNX5422;    -   Murine double minute (mdm2) oncogene inhibitors, such as        DS-3032b, RG7775, AMG-232, HDM201, and idasanutlin (RG7388);    -   CD137 agonists, such as urelumab, utomilumab (PF-05082566);    -   STING agonists, such as ADU-S100 (MIW-815), SB-11285, MK-1454,        SR-8291, AdVCA0848, GSK-532, SYN-STING, MSA-1, SR-8291;    -   FGFR inhibitors, such as FGF-401, INCB-054828, BAY-1163877,        AZD4547, JNJ-42756493, LY2874455, Debio-1347;    -   fatty acid synthase (FASN) inhibitors, such as TVB-2640;    -   Anti-KIR monoclonal antibodies, such as lirilumab (IPH-2102),        IPH-4102;    -   Antigen CD19 inhibitors, such as MOR208, MEDI-551, AFM-11,        inebilizumab;    -   CD44 binders, such as A6;    -   protein phosphatease 2A (PP2A) inhibitors, such as LB-100;    -   CYP17 inhibitors, such as seviteronel (VT-464), ASN-001,        ODM-204, CFG920, abiraterone acetate;    -   RXR agonists, such as IRX4204;    -   hedgehog/smoothened (hh/Smo) antagonists, such as taladegib,        patidegib;    -   complement C3 modulators, such as Imprime PGG;    -   IL-15 agonists, such as ALT-803, NKTR-255, and hetIL-15;    -   EZH2 (enhancer of zeste homolog 2) inhibitors, such as        tazemetostat, CPI-1205, GSK-2816126;    -   Oncolytic viruses, such as pelareorep, CG-0070, MV-NIS therapy,        HSV-1716, DS-1647, VCN-01, ONCOS-102, TBI-1401, tasadenoturev        (DNX-2401), vocimagene amiretrorepvec, RP-1, CVA21, Celyvir,        LOAd-703, OBP-301;    -   DOT1L (histone methyltransferase) inhibitors, such as        pinometostat (EPZ-5676);    -   toxins such as Cholera toxin, ricin, Pseudomonas exotoxin,        Bordetella pertussis adenylate cyclase toxin, diphtheria toxin,        and caspase activators;    -   DNA plasmids, such as BC-819    -   PLK inhibitors of PLK 1, 2, and 3, such as volasertib (PLK1);    -   WEEl inhibitors, such as AZD-1775 (adavosertib);    -   Rho kinase (ROCK) inhibitors, such as AT13148, KD025;    -   ERK inhibitors, such as GDC-0994, LY3214996, MK-8353;    -   IAP inhibitors, such as ASTX660, debio-1143, birinapant,        APG-1387, LCL-161;    -   RNA polymerase inhibitors, such has lurbinectedin (PM-1183),        CX-5461;    -   Tubulin inhibitors, such as PM-184, BAL-101553 (lisavanbulin),        and OXI-4503, fluorapacin (AC-0001), plinabulin;    -   Toll-like receptor 4 (TL4) agonists, such as G100, GSK1795091,        and PEPA-10;    -   Elongation factor 1 alpha 2 inhibitors, such as plitidepsin;    -   CD95 inhibitors, such as APG-101, APO-010, asunercept;    -   WT1 inhibitors, such as DSP-7888;    -   splicing factor 3B subunit1 (SF3B1) inhibitors, such as H3B-8800    -   PDGFR alpha/KIT mutant-specific inhibitors such as BLU-285;    -   retinoid Z receptor gamma (RORγ) agonists, such as LYC-55716;    -   Inhibitors of myeloid cell leukemia sequence 1 (MCL1) apoptosis        regulator, such as AMG-176, AMG-397, 5-64315, AZD-5991, 483-LM,        A-1210477, UMI-77, JKY-5-037; and tetrahydronaphthalene        derivatives, including those described in WO2016033486,        WO2017147410 and WO2018183418.    -   Inhibitors of protein tyrosine phosphatase, non-receptor type 11        (PTPN11 or SHP2), such as TNO155 (SHP-099), RMC-4550, JAB-3068,        RMC-4630; Examples of SHP2 inhibitors include, but are not        limited to, those described in WO-2018172984 and WO-2017211303;    -   Hematopoietic Progenitor Kinase 1 (HPK1) inhibitors; Examples of        Hematopoietic Progenitor Kinase 1 (HPK1) inhibitors include, but        are not limited to, those described in WO-2018183956,        WO-2018183964, WO-2018167147, WO-2018183964, WO-2016205942,        WO-2018049214, WO-2018049200, WO-2018049191, WO-2018102366,        WO-2018049152 and WO-2016090300;    -   Apoptosis Signal-Regulating Kinase (ASK) Inhibitors: ASK        inhibitors include ASK1 inhibitors. Examples of ASK1 inhibitors        include, but are not limited to, those described in WO        2011/008709 (Gilead Sciences) and WO 2013/112741 (Gilead        Sciences).    -   Bruton's Tyrosine Kinase (BTK) Inhibitors: Examples of BTK        inhibitors include, but are not limited to,        (S)-6-amino-9-(1-(but-2-ynoyl)pyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-8(9H)-one,        acalabrutinib (ACP-196), BGB-3111, CB988, HM71224, ibrutinib,        M-2951 (evobrutinib), M7583, tirabrutinib (ONO-4059), PRN-1008,        spebrutinib (CC-292), TAK-020, vecabrutinib, ARQ-531, SHR-1459,        DTRMWXHS-12, TAS-5315    -   Cluster of Differentiation 47 (CD47) inhibitors: Examples of        CD47 inhibitors include, but are not limited to anti-CD47 mAbs        (Vx-1004), anti-human CD47 mAbs (CNTO-7108), CC-90002,        CC-90002-ST-001, humanized anti-CD47 antibody (Hu5F9-G4),        NI-1701, NI-1801, RCT-1938, and TTI-621.    -   Cyclin-dependent Kinase (CDK) Inhibitors: CDK inhibitors include        inhibitors of CDK 1, 2, 3, 4, 6, 7 and 9, such as abemaciclib,        alvocidib (HMR-1275, flavopiridol), AT-7519, dinaciclib,        ibrance, FLX-925, LEE001, palbociclib, ribociclib, rigosertib,        selinexor, UCN-01, SY1365, CT-7001, SY-1365, G1T38, milciclib,        trilaciclib, and TG-02.    -   Discoidin Domain Receptor (DDR) Inhibitors: DDR inhibitors        include inhibitors of DDR1 and/or DDR2. Examples of DDR        inhibitors include, but are not limited to, those disclosed in        WO 2014/047624 (Gilead Sciences), US 2009-0142345 (Takeda        Pharmaceutical), US 2011-0287011 (Oncomed Pharmaceuticals), WO        2013/027802 (Chugai Pharmaceutical), and WO 2013/034933        (Imperial Innovations).    -   Histone Deacetylase (HDAC) Inhibitors: Examples of HDAC        inhibitors include, but are not limited to, abexinostat,        ACY-241, AR-42, BEBT-908, belinostat, CKD-581, CS-055        (HBI-8000), CUDC-907 (fimepinostat), entinostat, givinostat,        mocetinostat, panobinostat, pracinostat, quisinostat        (JNJ-26481585), resminostat, ricolinostat, SHIP-141, valproic        acid (VAL-001), vorinostat, tinostamustine, remetinostat,        entinostat.    -   Indoleamine-pyrrole-2,3-dioxygenase (IDO1) inhibitors: Examples        of IDO1 inhibitors include, but are not limited to, BLV-0801,        epacadostat, F-001287, GBV-1012, GBV-1028, GDC-0919, indoximod,        NKTR-218, NLG-919-based vaccine, PF-06840003,        pyranonaphthoquinone derivatives (SN-35837), resminostat,        SBLK-200802, BMS-986205, and shIDO-ST, EOS-200271, KHK-2455,        LY-3381916.    -   Janus Kinase (JAK) Inhibitors: JAK inhibitors inhibit JAK1,        JAK2, and/or JAK3. Examples of JAK inhibitors include, but are        not limited to, AT9283, AZD1480, baricitinib, BMS-911543,        fedratinib, filgotinib (GLPG0634), gandotinib (LY2784544),        INCB039110 (itacitinib), lestaurtinib, momelotinib (CYT0387),        NS-018, pacritinib (SB1518), peficitinib (ASP015K), ruxolitinib,        tofacitinib (formerly tasocitinib), INCB052793, and XL019.    -   Lysyl Oxidase-Like Protein (LOXL) Inhibitors: LOXL inhibitors        include inhibitors of LOXL1, LOXL2, LOXL3, LOXL4, and/or LOXL5.        Examples of LOXL inhibitors include, but are not limited to, the        antibodies described in WO 2009/017833 (Arresto Biosciences).        Examples of LOXL2 inhibitors include, but are not limited to,        the antibodies described in WO 2009/017833 (Arresto        Biosciences), WO 2009/035791 (Arresto Biosciences), and WO        2011/097513 (Gilead Biologics).    -   Matrix Metalloprotease (MMP) Inhibitors: MMP inhibitors include        inhibitors of MMP1 through 10. Examples of MMP9 inhibitors        include, but are not limited to, marimastat (BB-2516),        cipemastat (Ro 32-3555), GS-5745 (andecaliximab) and those        described in WO 2012/027721 (Gilead Biologics).    -   Mitogen-activated Protein Kinase (MEK) Inhibitors: MEK        inhibitors include antroquinonol, binimetinib, cobimetinib        (GDC-0973, XL-518), MT-144, selumetinib (AZD6244), sorafenib,        trametinib (GSK1120212), uprosertib+trametinib, PD-0325901,        pimasertib, LTT462, AS703988, CC-90003, refametinib.    -   Phosphatidylinositol 3-kinase (PI3K) Inhibitors: PI3K inhibitors        include inhibitors of PI3K7, PI3Kδ, PI3Kβ, PI3Kα, and/or        pan-PI3K. Examples of PI3K inhibitors include, but are not        limited to, ACP-319, AEZA-129, AMG-319, AS252424, AZD8186, BAY        10824391, BEZ235, buparlisib (BKM120), BYL719 (alpelisib),        CH5132799, copanlisib (BAY 80-6946), duvelisib, GDC-0032,        GDC-0077, GDC-0941, GDC-0980, GSK2636771, GSK2269557, idelalisib        (Zydelig®), INCB50465, IPI-145, IPI-443, IPI-549, KAR4141,        LY294002, LY3023414, MLN1117, OXY111A, PA799, PX-866, RG7604,        rigosertib, RP5090, RP6530, SRX3177, taselisib, TG100115,        TGR-1202 (umbralisib), TGX221, WX-037, X-339, X-414, XL147        (SAR245408), XL499, XL756, wortmannin, ZSTK474, and the        compounds described in WO 2005/113556 (ICOS), WO 2013/052699        (Gilead Calistoga), WO 2013/116562 (Gilead Calistoga), WO        2014/100765 (Gilead Calistoga), WO 2014/100767 (Gilead        Calistoga), and WO 2014/201409 (Gilead Sciences).    -   Tyrosine-Kinase Inhibitors (TKIs): TKIs may target epidermal        growth factor receptors (EGFRs) and receptors for fibroblast        growth factor (FGF), platelet-derived growth factor (PDGF), and        vascular endothelial growth factor (VEGF). Examples of TKIs        include, but are not limited to, afatinib, ARQ-087        (derazantinib), asp5878, AZD3759, AZD4547, bosutinib,        brigatinib, cabozantinib, cediranib, crenolanib, dacomitinib,        dasatinib, dovitinib, E-6201, erdafitinib, erlotinib, gefitinib,        gilteritinib (ASP-2215), FP-1039, HM61713, icotinib, imatinib,        KX2-391 (Src), lapatinib, lestaurtinib, lenvatinib, midostaurin,        nintedanib, ODM-203, osimertinib (AZD-9291), ponatinib,        poziotinib, quizartinib, radotinib, rociletinib, sulfatinib        (HMPL-012), sunitinib, famitinib L-malate, (MAC-4), tivoanib,        TH-4000, and MEDI-575 (anti-PDGFR antibody). TKIs may alos        target Spleen Tyrosine Kinase (SYK). Examples of SYK inhibitors        include, but are not limited to,        6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine,        BAY-61-3606, cerdulatinib (PRT-062607), entospletinib,        fostamatinib (R788), HMPL-523, NVP-QAB 205 AA, R112, R343,        tamatinib (R406), and those described in U.S. Pat. No. 8,450,321        (Gilead Connecticut) and those described in U.S. 2015/0175616.

Toll-Like Receptor (TLR) Modulators

In one embodiment, the additional therapeutic agent is a TLR modulator.TLR modulators may include modulators of TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13.

In certain embodiments, the antibodies or antigen-binding fragmentsdescribed herein are combined with a TLR agonist. Examples of TLRagonists include without limitation: vesatolimod (GS-9620), lefitolimod,tilsotolimod, rintatolimod, DSP-0509, AL-034, G-100, cobitolimod,AST-008, motolimod, GSK-1795091, GSK-2245035, VTX-1463, GS-9688,LHC-165, BDB-001, RG-7854, telratolimod. Additional examples include butare not limited to the compounds described in U.S. Pat. No. 8,367,670and the compounds described in U.S. Patent Application Publication No.2016/0289229. In one embodiment, the antibody of the present inventionmay be combined with TLR7 agonist such as Vesatolimod. In anotherembodiment, the antibody of the present invention may be combined withTLR8 agonist.

Examples of TLR3 modulators include rintatolimod, poly-ICLC, RIBOXXON®,Apoxxim, RIBOXXIM®, IPH-33, MCT-465, MCT-475, and ND-1.1. Examples ofTLR7 modulators include GS-9620, GSK-2245035, imiquimod, resiquimod,DSR-6434, DSP-3025, IMO-4200, MCT-465, MEDI-9197, 3M-051, SB-9922,3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7795, and the compoundsdisclosed in US20100143301 (Gilead Sciences), US20110098248 (GileadSciences), and US20090047249 (Gilead Sciences). Examples of TLR8modulators include motolimod, resiquimod, 3M-051, 3M-052, MCT-465,IMO-4200, VTX-763, VTX-1463, and the compounds disclosed inUS20140045849 (Janssen), US20140073642 (Janssen), WO2014/056953(Janssen), WO2014/076221 (Janssen), WO2014/128189 (Janssen),US20140350031 (Janssen), WO2014/023813 (Janssen), US20080234251 (ArrayBiopharma), US20080306050 (Array Biopharma), US20100029585 (VentirxPharma), US20110092485 (Ventirx Pharma), US20110118235 (Ventirx Pharma),US20120082658 (Ventirx Pharma), US20120219615 (Ventirx Pharma),US20140066432 (Ventirx Pharma), US20140088085 (Ventirx Pharma),US20140275167 (Novira Therapeutics), and US20130251673 (NoviraTherapeutics). Examples of TLR9 modulators include BB-001, BB-006,CYT-003, IMO-2055, IMO-2125, IMO-3100, IMO-8400, IR-103, IMO-9200,agatolimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, leftolimod(MGN-1703), litenimod, and CYT-003-QbG10.

Examples of TLR8 inhibitors include, but are not limited to, E-6887,IMO-4200, IMO-8400, IMO-9200, MCT-465, MEDI-9197, motolimod, resiquimod,GS-9688, VTX-1463, and VTX-763.

Examples of TLR9 inhibitors include, but are not limited to, AST-008,IMO-2055, IMO-2125, lefitolimod, litenimod, MGN-1601, and PUL-042.

Chemotherapeutic Agents

i.e. Examples of chemotherapeutic agents include but not limited to:alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan, and piposulfan;aziridines such as benzodepa, carboquone, meturedepa, and uredepa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimemylolomelamine; acetogenins,especially bullatacin and bullatacinone; a camptothecin, includingsynthetic analog topotecan; bryostatin, callystatin; CC-1065, includingits adozelesin, carzelesin, and bizelesin synthetic analogs;cryptophycins, particularly cryptophycin 1 and cryptophycin 8;dolastatin; duocarmycin, including the synthetic analogs KW-2189 andCBI-TMI; eleutherobin; 5-azacytidine; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cyclophosphamide, glufosfamide, evofosfamide, bendamustine,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, and uracil mustard; nitrosoureas such as carmustine,chlorozotocin, foremustine, lomustine, nimustine, and ranimustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammaII and calicheamicin phiIl), dynemicinincluding dynemicin A, bisphosphonates such as clodronate, anesperamicin, neocarzinostatin chromophore and related chromoproteinenediyne antibiotic chromomophores, aclacinomycins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, carabicin,carrninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogs such asdemopterin, methotrexate, pteropterin, and trimetrexate; purine analogssuch as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, andfloxuridine; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, and testolactone; anti-adrenals such asaminoglutethimide, mitotane, and trilostane; folic acid replinisherssuch as frolinic acid; radiotherapeutic agents such as Radium-223;trichothecenes, especially T-2 toxin, verracurin A, roridin A, andanguidine; taxoids or taxanes such as paclitaxel (TAXOL®), abraxane,docetaxel (TAXOTERE®), cabazitaxel, BIND-014, tesetaxel; platinumanalogs such as cisplatin and carboplatin, NC-6004 nanoplatin;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;losoxantrone; fluoropyrimidine; folinic acid; podophyllinic acid;2-ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; trabectedin,triaziquone; 2,2′,2″-tricUorotriemylamine; urethane; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiopeta;chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine;methotrexate; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitroxantrone; vancristine; vinorelbine (NAVELBINE®); novantrone;teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate;CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DFMO); retinoids such as retinoic acid; capecitabine; NUC-1031; aFOLFOX (5-fluorouracil, leucovorin, oxaliplatin) regimen (e.g.,FOLFOX-4, FOLFOX-6, modified FOLFOX-6 (mFOLFOX-6), FOLFOX-7), FOLFIRI(fluorouracil, leucovorin, and irinotecan); and pharmaceuticallyacceptable salts, acids, or derivatives of any of the above.

Anti-Hormonal Agents

Examples of anti-estrogens and SERMs include, for example, tamoxifen(including NOLVADEX™), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene(FARESTON®).

Inhibitors of the enzyme aromatase regulate estrogen production in theadrenal glands. Examples include 4(5)-imidazoles, aminoglutethimide,megestrol acetate (MEGACE®), exemestane, formestane, fadrozole, vorozole(RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®).

Examples of anti-androgens include apalutamide, abiraterone,enzalutamide, flutamide, galeterone, nilutamide, bicalutamide,leuprolide, goserelin, ODM-201, APC-100, ODM-204.

Examples of progesterone receptor antagonists include onapristone.

Anti-Angiogenic Agents

Anti-angiogenic agents include, but are not limited to, retinoid acidand derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN®, ENDOSTATIN®,regorafenib, necuparanib, suramin, squalamine, tissue inhibitor ofmetalloproteinase-1, tissue inhibitor of metalloproteinase-2,plasminogen activator inhibitor-1, plasminogen activator inbibitor-2,cartilage-derived inhibitor, paclitaxel (nab-paclitaxel), plateletfactor 4, protamine sulphate (clupeine), sulphated chitin derivatives(prepared from queen crab shells), sulphated polysaccharidepeptidoglycan complex (sp-pg), staurosporine, modulators of matrixmetabolism including proline analogs such as 1-azetidine-2-carboxylicacid (LACA), cishydroxyproline, d,l-3,4-dehydroproline, thiaproline,α,α′-dipyridyl, beta-aminopropionitrile fumarate,4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone, methotrexate, mitoxantrone,heparin, interferons, 2 macroglobulin-serum, chicken inhibitor ofmetalloproteinase-3 (ChIMP-3), chymostatin, beta-cyclodextrintetradecasulfate, eponemycin, fumagillin, gold sodium thiomalate,d-penicillamine, beta-1-anticollagenase-serum, alpha-2-antiplasmin,bisantrene, lobenzarit disodium, n-2-carboxyphenyl-4-chloroanthronilicacid disodium or “CCA”, thalidomide, angiostatic steroid, carboxyaminoimidazole, metalloproteinase inhibitors such as BB-94, inhibitorsof S100A9 such as tasquinimod. Other anti-angiogenesis agents includeantibodies, preferably monoclonal antibodies against these angiogenicgrowth factors: beta-FGF, alpha-FGF, FGF-5, VEGF isoforms, VEGF-C,HGF/SF, and Ang-1/Ang-2.

Anti-Fibrotic Agents

Anti-fibrotic agents include, but are not limited to, the compounds suchas beta-aminoproprionitrile (BAPN), as well as the compounds disclosedin U.S. Pat. No. 4,965,288 relating to inhibitors of lysyl oxidase andtheir use in the treatment of diseases and conditions associated withthe abnormal deposition of collagen and U.S. Pat. No. 4,997,854 relatingto compounds which inhibit LOX for the treatment of various pathologicalfibrotic states, which are herein incorporated by reference. Furtherexemplary inhibitors are described in U.S. Pat. No. 4,943,593 relatingto compounds such as 2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine,U.S. Pat. Nos. 5,021,456, 5,059,714, 5,120,764, 5,182,297, 5,252,608relating to 2-(1-naphthyloxymemyl)-3-fluoroallylamine, and US2004-0248871, which are herein incorporated by reference.

Exemplary anti-fibrotic agents also include the primary amines reactingwith the carbonyl group of the active site of the lysyl oxidases, andmore particularly those which produce, after binding with the carbonyl,a product stabilized by resonance, such as the following primary amines:emylenemamine, hydrazine, phenylhydrazine, and their derivatives;semicarbazide and urea derivatives; aminonitriles such as BAPN or2-nitroethylamine; unsaturated or saturated haloamines such as2-bromo-ethylamine, 2-chloroethylamine, 2-trifluoroethylamine,3-bromopropylamine, and p-halobenzylamines; and selenohomocysteinelactone.

Other anti-fibrotic agents are copper chelating agents penetrating ornot penetrating the cells. Exemplary compounds include indirectinhibitors which block the aldehyde derivatives originating from theoxidative deamination of the lysyl and hydroxylysyl residues by thelysyl oxidases. Examples include the thiolamines, particularlyD-penicillamine, and its analogs such as2-amino-5-mercapto-5-methylhexanoic acid,D-2-amino-3-methyl-3-((2-acetamidoethyl)dithio)butanoic acid,p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid,sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butane sulphurate,2-acetamidoethyl-2-acetamidoethanethiol sulphanate, andsodium-4-mercaptobutanesulphinate trihydrate.

Immunotherapeutic Agents

The immunotherapeutic agents include and are not limited to therapeuticantibodies suitable for treating patients. Some examples of therapeuticantibodies include abagovomab, ABP-980, adecatumumab, afutuzumab,alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab,bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab,brentuximab, cantuzumab, catumaxomab, CC49, cetuximab, citatuzumab,cixutumumab, clivatuzumab, conatumumab, dacetuzumab, dalotuzumab,daratumumab, detumomab, dinutuximab, drozitumab, duligotumab,dusigitumab, ecromeximab, elotuzumab, emibetuzumab, ensituximab,ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab,flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab,glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab,inotuzumab, intetumumab, ipilimumab (YERVOY®, MDX-010, BMS-734016, andMDX-101), iratumumab, labetuzumab, lexatumumab, lintuzumab,lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab,minretumomab, mitumomab, mogamulizumab, moxetumomab, naptumomab,narnatumab, necitumumab, nimotuzumab, nofetumomab, OBI-833,obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab,oportuzumab, oregovomab, panitumumab, parsatuzumab, pasudotox,patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab,radretumab, ramucirumab (Cyramza®), rilotumumab, rituximab, robatumumab,samalizumab, satumomab, sibrotuzumab, siltuximab, solitomab, simtuzumab,tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab,tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab,vorsetuzumab, votumumab, zalutumumab, and 3F8.

The exemplified therapeutic antibodies may be further labeled orcombined with a radioisotope particle such as indium-111, yttrium-90, oriodine-131.

Cancer Gene Therapy and Cell Therapy

Cancer Gene Therapy and Cell Therapy include the insertion of a normalgene into cancer cells to replace a mutated or altered gene; geneticmodification to silence a mutated gene; genetic approaches to directlykill the cancer cells; including the infusion of immune cells designedto replace most of the patient's own immune system to enhance the immuneresponse to cancer cells, or activate the patient's own immune system (Tcells or Natural Killer cells) to kill cancer cells, or find and killthe cancer cells; genetic approaches to modify cellular activity tofurther alter endogenous immune responsiveness against cancer.

Gene Editors

The genome editing system is selected from the group consisting of: aCRISPR/Cas9 system, a zinc finger nuclease system, a TALEN system, ahoming endonucleases system, and a meganuclease system.

CAR-T Cell Therapy and TCR-T Cell Therapy

A population of immune effector cells engineered to express a chimericantigen receptor (CAR), wherein the CAR comprises a tumorantigen-binding domain. The immune effector cell is a T cell or an NKcell. TCR-T cells are engineered to target tumor derived peptidespresent on the surface of tumor cells. Cells can be autologous orallogeneic.

In some embodiments, the CAR comprises an antigen binding domain, atransmembrane domain, and an intracellular signalling domain.

In some embodiments, the intracellular domain comprises a primarysignaling domain, a costimulatory domain, or both of a primary signalingdomain and a costimulatory domain.

In some embodiments, the primary signaling domain comprises a functionalsignaling domain of one or more proteins selected from the groupconsisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcRgamma (FCERIG), FcR beta (Fc Epsilon Rlb), CD79a, CD79b, Fcgamma RIIa,DAP10, and DAP12.

In some embodiments, the costimulatory domain comprises a functionaldomain of one or more proteins selected from the group consisting ofCD27, CD28, 4-1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-I), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, CD4, CD8alpha, CD8beta,IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4,CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 1 ld, ITGAE, CD103, ITGAL, CD 1la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMFI, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D.

In some embodiments, the transmembrane domain comprises a transmembranedomain of a protein selected from the group consisting of the alpha,beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278),4-1BB(CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1),CD160, CD19, IL2Rbeta, IL2R gamma, IL7Ru, ITGA1, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CD1la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMFI, CD150, IPO-3), BLAME(SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, andNKG2C.

In some embodiments, the antigen binding domain binds a tumor antigen.In some embodiments, the tumor antigen is selected from the groupconsisting of: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to asCD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-likemolecule-1 (CLL-1 or CLECLI); CD33; epidermal growth factor receptorvariant III (EGFRvlll); ganglioside G2 (GD2); ganglioside GD3(aNeuSAc(2-8)aNeuSAc(2-3)bDGaip(1-4)bDGIcp(1-1)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GaINAcu-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (RORI); Fms-Like, Tyrosine Kinase3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6;Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule(EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunitalpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha(IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine21(Testisin or PRSS21); vascular endothelial growth factor receptor 2(VEGFR2); Lewis(Y)antigen; CD24; Platelet-derived growth factor receptorbeta (PDGFR-beta); Stage-specificembryonic antigen-4 (SSEA-4); CD20;delta like 3 (DLL3); Folate receptor alpha; Receptor tyrosine-proteinkinase, ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1);epidermal growth factor receptor (EGFR); neural cell adhesion molecule(NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP);insulin-like growth factor 1 receptor (IGF-I receptor); carbonicanhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type,9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consistingof breakpoint cluster region (BCR) and Abelson murineleukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor2(EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); gangliosideGM3 (aNeuSAc(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanomaassociatedantigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1(TEM1/CD248); tumor endothelial marker 7-related (TEM7R); sixtransmembrane epithelial antigen of the prostate I (STEAP1); claudin 6(CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupledreceptor class C group 5, member D (GPRCSD); chromosome X open readingframe 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK);Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion ofgloboH glycoceramide (GloboH); mammary gland differentiation antigen(NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1(HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); Gprotein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locusK 9 (LY6K); Olfactory receptor 51E2 (ORS IE2); TCR Gamma AlternateReading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testisantigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-la);Melanomaassociated antigen 1 (MAGE-A1); ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, Member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MADCT-1); melanomacancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor proteinp53, (p53); p53 mutant; prostein; survivin; telomerase; prostatecarcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigenrecognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant;human Telomerase reverse transcriptase (hTERT); sarcoma translocationbreakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viraloncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family MemberC (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1(CYPIBI); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brotherof the Regulator of Imprinted Sites), Squamous Cell Carcinoma AntigenRecognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5);proacrosin binding protein sp32 (OY-TES I); lymphocyte-specific proteintyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovialsarcoma, X breakpoint 2 (SSX2); Receptor for Advanced GlycationEndproducts (RAGE-I); renal ubiquitous 1 (RUI); renal ubiquitous 2(RU2); legumain; human papilloma virus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associatedimmunoglobulin-like receptor 1 (LAIRI); Fc fragment of IgA receptor(FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily Amember 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-typelectin domain family 12 member A (CLEC12A); bone marrow stromal cellantigen 2 (BST2); EGF-like module containing mucin-like hormonereceptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3);Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1(IGLL1).

In some embodiments, the tumor antigen is selected from CD150, 5T4,ActRIIA, B7, BMCA, CA-125, CCNA1, CD123, CD126, CD138, CD14, CD148,CD15, CD19, CD20, CD200, CD21, CD22, CD23, CD24, CD25, CD26, CD261,CD262, CD30, CD33, CD362, CD37, CD38, CD4, CD40, CD40L, CD44, CD46, CD5,CD52, CD53, CD54, CD56, CD66a-d, CD74, CD8, CD80, CD92, CE7, CS-1,CSPG4, ED-B fibronectin, EGFR, EGFRvIII, EGP-2, EGP-4, EPHa2, ErbB2,ErbB3, ErbB4, FBP, GD2, GD3, HER1-HER2 in combination, HER2-HER3 incombination, HERV-K, HIV-1 envelope glycoprotein gp120, HIV-1 envelopeglycoprotein gp41, HLA-DR, HM1.24, HMW-MAA, Her2, Her2/neu, IGF-1R,IL-11Ralpha, IL-13R-alpha2, IL-2, IL-22R-alpha, IL-6, IL-6R, Ia, Ii,L1-CAM, L1-cell adhesion molecule, Lewis Y, Ll-CAM, MAGE A3, MAGE-A1,MART-1, MUC1, NKG2C ligands, NKG2D Ligands, NYESO-1, OEPHa2, PIGF, PSCA,PSMA, ROR1, T101, TAC, TAG72, TIM-3, TRAIL-R1, TRAIL-R1 (DR4), TRAIL-R2(DR5), VEGF, VEGFR2, WT-I, a G-protein coupled receptor,alphafetoprotein (AFP), an angiogenesis factor, an exogenous cognatebinding molecule (ExoCBM), oncogene product, anti-folate receptor,c-Met, carcinoembryonic antigen (CEA), cyclin (D 1), ephrinB2,epithelial tumor antigen, estrogen receptor, fetal acethycholine ereceptor, folate binding protein, gp100, hepatitis B surface antigen,kappa chain, kappa light chain, kdr, lambda chain, livin,melanoma-associated antigen, mesothelin, mouse double minute 2 homolog(MDM2), mucin 16 (MUC16), mutated p53, mutated ras, necrosis antigens,oncofetal antigen, ROR2, progesterone receptor, prostate specificantigen, tEGFR, tenascin, P2-Microgiobuiin, Fc Receptor-like 5 (FcRL5).

Non limiting examples of cell therapies are: Algenpantucel-L,Sipuleucel-T, (BPX-501) rivogenlecleucel U.S. Pat. No. 9,089,520,WO2016100236, AU-105, ACTR-087, activated allogeneic natural killercells CNDO-109-AANK, MG-4101, AU-101, BPX-601, FATE-NK100, LFU-835hematopoietic stem cells, Imilecleucel-T, baltaleucel-T, PNK-007,UCARTCS1, ET-1504, ET-1501, ET-1502, ET-190, CD19-ARTEMIS, ProHema,FT-1050-treated bone marrow stem cell therapy, CD4CARNK-92 cells,CryoStim, AlloStim, lentiviral transduced huCART-meso cells, CART-22cells, EGFRt/19-28z/4-1BBL CAR T cells, autologous 4H11-28z/fIL-12/EFGRtT cell, CCR5-SBC-728-HSPC, CAR4-1BBZ, CH-296, dnTGFbRII-NY-ESOc259T,Ad-RTS-IL-12, IMA-101, IMA-201, CARMA-0508, TT-18, CMD-501, CMD-503,CMD-504, CMD-502, CMD-601, CMD-602, CSG-005.

In certain embodiments, the cell therapy comprises an agent targeting:Alpha-fetoprotein, such as ET-1402, and AFP-TCR; Anthrax toxin receptor1, such as anti-TEM8 CAR T-cell therapy; B cell maturation antigens(BCMA), such as bb-2121, UCART-BCMA, ET-140, KITE-585, MCM-998,LCAR-B38M, CART-BCMA, SEA-BCMA, BB212, UCART-BCMA, ET-140, P-BCMA-101,and AUTO-2 (APRIL-CAR); Anti-CLL-1 antibodies, such as KITE-796;Anti-PD-L1-CAR tank cell therapy, such as KD-045; B7 homolog 6, such asCAR-NKp30 and CAR-B7H6; B-lymphocyte antigen CD19, such as TBI-1501,CTL-119 huCART-19 T cells, JCAR-015 U.S. Pat. No. 7,446,190, JCAR-014,JCAR-017, (WO2016196388, WO2016033570, WO2015157386), axicabtageneciloleucel (KTE-C19), U.S. Pat. Nos. 7,741,465, 6,319,494, UCART-19,EBV-CTL, T tisagenlecleucel-T (CTL019), WO2012079000, WO2017049166,CD19CAR-CD28-CD3zeta-EGFRt-expressing T cells, CD19/4-1BBL armored CAR Tcell therapy, C-CAR-011, CIK-CAR.CD19, CD19CAR-28-zeta T cells,PCAR-019, MatchCART, DSCAR-01, and IM19 CAR-T; B-lymphocyte antigenCD20, such as ACTR707+Rituximab; B-lymphocyte antigen CD22, such asJCAR-018 (see WO2016090190); NY-ESO-1, such as GSK-3377794 and TBI-1301;Carbonic anhydrase, such as DC-Ad-GMCAIX; Caspase 9 suicide gene, suchas CaspaCIDe DLI and BPX-501; CCR5, such as SB-728; CDw123, such asMB-102 and UCART-123; CD20m such as CBM-C20.1; CD22 such as UCART-22;CD4, such as ICG-122; CD30, such as CART30 (CBM-C30.1; CD33, such asCIK-CAR.CD33; CD38, such as T-007, UCART-38; CD40 ligand, such asBPX-201; CEACAM protein 5 modulators, such as MG7-CART; Claudin 6, suchas CSG-002; EBV, such as CMD-003; MUC16, such as autologous4H11-28z/fIL-12/EFGRt T cell; Endonuclease, such as PGN-514 and PGN-201;Epstein-Barr virus specific T-lymphocytes, such as TT-10; Ganglioside(GD2), such as 4SCAR-GD2; Glutamate carboxypeptidase II, such asCIK-CAR.PSMA, CART-PSMA-TGFβRDN, P-PSMA-101; Hemoglobin, such asPGN-236; Hepatocyte growth factor receptor, such as anti-cMet RNA CAR T;Human papillomavirus E7 protein, such as KITE-439; Immunoglobulin gammaFc receptor III, such as ACTR087; IL-12, such as DC-RTS-IL-12; IL-12agonist/mucin 16, such as JCAR-020; IL-13 alpha 2, such as MB-101; K-RasGTPase, such as anti-KRAS G12V mTCR cell therapy; Neural cell adhesionmolecule L1 L1CAM (CD171), such as JCAR-023; Latent membrane protein1/Latent membrane protein 2, such as Ad5f35-LMPd1-2-transducedautologous dendritic cells; Melanoma associated antigen 10, such asMAGE-A10C796T MAGE-A10 TCR; Melanoma associated antigen 3/Melanomaassociated antigen 6 (MAGE A3/A6) such as KITE-718; Mesothelin, such asCSG-MESO, TC-210; NKG2D, such as NKR-2; Ntrkrl tyrosine kinase receptor,such as JCAR-024; PRAME, such as BPX-701; gp100 antigen, such asIMCgp100; Wilms tumor protein, such as JTCR-016 and WT1-CTL.

Types of Cancer

In some embodiments, the cancer is Burkitt's lymphoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma (NHL), indolent non-Hodgkin's lymphoma(iNHL), refractory iNHL, multiple myeloma (MM), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), B-cell ALL, acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma (SLL), myelodysplastic syndrome (MDS), myeloproliferativedisease (MPD), mantle cell lymphoma (MCL), follicular lymphoma (FL),Waldestrom's macroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma,diffuse large B-cell lymphoma (DLBCL), or marginal zone lymphoma (MZL).In one embodiment, the cancer is minimal residual disease (MRD). Inadditional embodiment, the cancer is selected from Hodgkin's lymphoma,non-Hodgkin's lymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL),and refractory iNHL. In certain embodiment, the cancer is indolentnon-Hodgkin's lymphoma (iNHL). In some embodiment, the cancer isrefractory iNHL. In one embodiment, the cancer is chronic lymphocyticleukemia (CLL). In other embodiment, the cancer is diffuse large B-celllymphoma (DLBCL).

In certain embodiments, the cancer is a solid tumor is selected from thegroup consisting of pancreatic cancer; bladder cancer; colorectalcancer; breast cancer, including metastatic breast cancer; prostatecancer, including androgen-dependent and androgen-independent prostatecancer; kidney or renal cancer, including, e.g., metastatic renal cellcarcinoma; hepatocellular cancer; lung cancer, including, e.g.,non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC),and adenocarcinoma of the lung; ovarian cancer, including, e.g.,progressive epithelial or primary peritoneal cancer; cervical cancer;gastric cancer; esophageal cancer; head and neck cancer, including,e.g., squamous cell carcinoma of the head and neck; melanoma;neuroendocrine cancer, including metastatic neuroendocrine tumors; braintumors, including, e.g., glioma, anaplastic oligodendroglioma, adultglioblastoma multiforme, and adult anaplastic astrocytoma; bone cancer;and soft tissue sarcoma, hepatic carcinoma, rectal cancer, penilecarcinoma, vulval cancer, thyroid cancer, salivary gland carcinoma,endometrial or uterine carcinoma, hepatoma, hepatocellular cancer, livercancer, gastric or stomach cancer including gastrointestinal cancer,cancer of the peritoneum, squamous carcinoma of the lung,gastroesophagal cancer, biliary tract cancer, gall bladder cancer,colorectal/appendiceal cancer, squamous cell cancer (e.g., epithelialsquamous cell cancer).

Any of the methods of treatment provided may be used to treat cancer atvarious stages. By way of example, the cancer stage includes but is notlimited to early, advanced, locally advanced, remission, refractory,reoccurred after remission and progressive.

Subjects

Any of the methods of treatment provided may be used to treat a subject(e.g., human) who has been diagnosed with or is suspected of havingcancer. As used herein, a subject refers to a mammal, including, forexample, a human.

In some embodiments, the subject may be a human who exhibits one or moresymptoms associated with cancer or hyperproliferative disease. In someembodiments, the subject may be a human who exhibits one or moresymptoms associated with cancer. In some embodiments, the subject is atan early stage of a cancer. In other embodiments, the subject is at anadvanced stage of cancer.

In certain, the subject may be a human who is at risk, or genetically orotherwise predisposed (e.g., risk factor) to developing cancer orhyperproliferative disease who has or has not been diagnosed. As usedherein, an “at risk” subject is a subject who is at risk of developingcancer. The subject may or may not have detectable disease, and may ormay not have displayed detectable disease prior to the treatment methodsdescribed herein. An at risk subject may have one or more so-called riskfactors, which are measurable parameters that correlate with developmentof cancer, which are described herein. A subject having one or more ofthese risk factors has a higher probability of developing cancer than anindividual without these risk factor(s). These risk factors may include,for example, age, sex, race, diet, history of previous disease, presenceof precursor disease, genetic (e.g., hereditary) considerations, andenvironmental exposure. In some embodiments, the subjects at risk forcancer include, for example, those having relatives who have experiencedthe disease, and those whose risk is determined by analysis of geneticor biochemical markers.

In addition, the subject may be a human who is undergoing one or morestandard therapies, such as chemotherapy, radiotherapy, immunotherapy,surgery, or combination thereof. Accordingly, one or more kinaseinhibitors may be administered before, during, or after administrationof chemotherapy, radiotherapy, immunotherapy, surgery or combinationthereof.

In certain embodiments, the subject may be a human who is (i)substantially refractory to at least one chemotherapy treatment, or (ii)is in relapse after treatment with chemotherapy, or both (i) and (ii).In some of embodiments, the subject is refractory to at least two, atleast three, or at least four chemotherapy treatments (includingstandard or experimental chemotherapies).

Lymphoma or Leukemia Combination Therapy

Some chemotherapy agents are suitable for treating lymphoma or leukemia.These agents include aldesleukin, alvocidib, amifostine trihydrate,aminocamptothecin, antineoplaston A10, antineoplaston AS2-1,anti-thymocyte globulin, arsenic trioxide, Bcl-2 family proteininhibitor ABT-263, beta alethine, BMS-345541, bortezomib (VELCADE®),bortezomib (VELCADE®, PS-341), bryostatin 1, bulsulfan, campath-1H,carboplatin, carfilzomib (Kyprolis®), carmustine, caspofungin acetate,CC-5103, chlorambucil, CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone), cisplatin, cladribine, clofarabine, curcumin, CVP(cyclophosphamide, vincristine, and prednisone), cyclophosphamide,cyclosporine, cytarabine, denileukin diftitox, dexamethasone, docetaxel,dolastatin 10, doxorubicin, doxorubicin hydrochloride, DT-PACE(dexamethasone, thalidomide, cisplatin, doxorubicin, cyclophosphamide,and etoposide), enzastaurin, epoetin alfa, etoposide, everolimus(RAD001), FCM (fludarabine, cyclophosphamide, and mitoxantrone), FCR(fludarabine, cyclophosphamide, and rituximab), fenretinide, filgrastim,flavopiridol, fludarabine, FR (fludarabine and rituximab), geldanamycin(17 AAG), hyperCVAD (hyperfractionated cyclophosphamide, vincristine,doxorubicin, dexamethasone, methotrexate, and cytarabine), ICE(iphosphamide, carboplatin, and etoposide), ifosfamide, irinotecanhydrochloride, interferon alpha-2b, ixabepilone, lenalidomide(REVLIMID®, CC-5013), lymphokine-activated killer cells, MCP(mitoxantrone, chlorambucil, and prednisolone), melphalan, mesna,methotrexate, mitoxantrone hydrochloride, motexafin gadolinium,mycophenolate mofetil, nelarabine, obatoclax (GX15-070), oblimersen,octreotide acetate, omega-3 fatty acids, Omr-IgG-am (WNIG, Omrix),oxaliplatin, paclitaxel, palbociclib (PD0332991), pegfilgrastim,PEGylated liposomal doxorubicin hydrochloride, perifosin, prednisolone,prednisone, recombinant flt3 ligand, recombinant human thrombopoietin,recombinant interferon alfa, recombinant interleukin-11, recombinantinterleukin-12, rituximab, R-CHOP (rituximab and CHOP), R-CVP (rituximaband CVP), R-FCM (rituximab and FCM), R-ICE (rituximab and ICE), and RMCP (rituximab and MCP), R-roscovitine (seliciclib, CYC202),sargramostim, sildenafil citrate, simvastatin, sirolimus, styrylsulphones, tacrolimus, tanespimycin, temsirolimus (CCl-779),thalidomide, therapeutic allogeneic lymphocytes, thiotepa, tipifarnib,vincristine, vincristine sulfate, vinorelbine ditartrate, SAHA(suberanilohydroxamic acid, or suberoyl, anilide, and hydroxamic acid),vemurafenib (Zelboraf®), venetoclax (ABT-199).

One modified approach is radioimmunotherapy, wherein a monoclonalantibody is combined with a radioisotope particle, such as indium-111,yttrium-90, and iodine-131. Examples of combination therapies include,but are not limited to, iodine-131 tositumomab (BEXXAR®), yttrium-90ibritumomab tiuxetan (ZEVALIN®), and BEXXAR® with CHOP.

The abovementioned therapies can be supplemented or combined with stemcell transplantation or treatment. Therapeutic procedures includeperipheral blood stem cell transplantation, autologous hematopoieticstem cell transplantation, autologous bone marrow transplantation,antibody therapy, biological therapy, enzyme inhibitor therapy, totalbody irradiation, infusion of stem cells, bone marrow ablation with stemcell support, in vitro-treated peripheral blood stem celltransplantation, umbilical cord blood transplantation, immunoenzymetechnique, low-LET cobalt-60 gamma ray therapy, bleomycin, conventionalsurgery, radiation therapy, and nonmyeloablative allogeneichematopoietic stem cell transplantation.

Non-Hodgkin's Lymphomas Combination Therapy

Treatment of non-Hodgkin's lymphomas (NHL), especially those of B cellorigin, includes using monoclonal antibodies, standard chemotherapyapproaches (e.g., CHOP, CVP, FCM, MCP, and the like),radioimmunotherapy, and combinations thereof, especially integration ofan antibody therapy with chemotherapy.

Examples of unconjugated monoclonal antibodies for the treatment ofNHL/B-cell cancers include rituximab, alemtuzumab, human or humanizedanti-CD20 antibodies, lumiliximab, anti-TNF-related apoptosis-inducingligand (anti-TRAIL), bevacizumab, galiximab, epratuzumab, SGN-40, andanti-CD74.

Examples of experimental antibody agents used in treatment of NHL/B-cellcancers include ofatumumab, ha20, PRO131921, alemtuzumab, galiximab,SGN-40, CHIR-12.12, epratuzumab, lumiliximab, apolizumab, milatuzumab,and bevacizumab.

Examples of standard regimens of chemotherapy for NHL/B-cell cancersinclude CHOP, FCM, CVP, MCP, R-CHOP, R-FCM, R-CVP, and R MCP.

Examples of radioimmunotherapy for NHL/B-cell cancers include yttrium-90ibritumomab tiuxetan (ZEVALIN®) and iodine-131 tositumomab (BEXXAR®).

Mantle Cell Lymphoma Combination Therapy

Therapeutic treatments for mantle cell lymphoma (MCL) includecombination chemotherapies such as CHOP, hyperCVAD, and FCM. Theseregimens can also be supplemented with the monoclonal antibody rituximabto form combination therapies R-CHOP, hyperCVAD-R, and R-FCM. Any of theabovementioned therapies may be combined with stem cell transplantationor ICE in order to treat MCL.

An alternative approach to treating MCL is immunotherapy. Oneimmunotherapy uses monoclonal antibodies like rituximab. Another usescancer vaccines, such as GTOP-99, which are based on the genetic makeupof an individual patient's tumor.

A modified approach to treat MCL is radioimmunotherapy, wherein amonoclonal antibody is combined with a radioisotope particle, such asiodine-131 tositumomab (BEXXAR®) and yttrium-90 ibritumomab tiuxetan(ZEVALIN®). In another example, BEXXAR® is used in sequential treatmentwith CHOP.

Other approaches to treating MCL include autologous stem celltransplantation coupled with high-dose chemotherapy, administeringproteasome inhibitors such as bortezomib (VELCADE® or PS-341), oradministering antiangiogenesis agents such as thalidomide, especially incombination with rituximab.

Another treatment approach is administering drugs that lead to thedegradation of Bcl-2 protein and increase cancer cell sensitivity tochemotherapy, such as oblimersen, in combination with otherchemotherapeutic agents.

A further treatment approach includes administering mTOR inhibitors,which can lead to inhibition of cell growth and even cell death.Non-limiting examples are sirolimus, temsirolimus (TORISEL®, CCI-779),CC-115, CC-223, SF-1126, PQR-309 (bimiralisib), voxtalisib, GSK-2126458,and temsirolimus in combination with RITUXAN®, VELCADE®, or otherchemotherapeutic agents.

Other recent therapies for MCL have been disclosed. Such examplesinclude flavopiridol, palbociclib (PD0332991), R-roscovitine(selicicilib, CYC202), styryl sulphones, obatoclax (GX15-070), TRAIL,Anti-TRAIL death receptors DR4 and DR5 antibodies, temsirolimus(TORISEL®, CCl-779), everolimus (RAD001), BMS-345541, curcumin, SAHA,thalidomide, lenalidomide (REVLIMID®, CC-5013), and geldanamycin (17AAG).

Waldenstrom's Macroglobulinemia Combination Therapy

Therapeutic agents used to treat Waldenstrom's Macroglobulinemia (WM)include aldesleukin, alemtuzumab, alvocidib, amifostine trihydrate,aminocamptothecin, antineoplaston A10, antineoplaston AS2-1,anti-thymocyte globulin, arsenic trioxide, autologous humantumor-derived HSPPC-96, Bcl-2 family protein inhibitor ABT-263, betaalethine, bortezomib (VELCADE®), bryostatin 1, busulfan, campath-1H,carboplatin, carmustine, caspofungin acetate, CC-5103, cisplatin,clofarabine, cyclophosphamide, cyclosporine, cytarabine, denileukindiftitox, dexamethasone, docetaxel, dolastatin 10, doxorubicinhydrochloride, DT-PACE, enzastaurin, epoetin alfa, epratuzumab(hLL2-anti-CD22 humanized antibody), etoposide, everolimus, fenretinide,filgrastim, fludarabine, ifosfamide, indium-111 monoclonal antibodyMN-14, iodine-131 tositumomab, irinotecan hydrochloride, ixabepilone,lymphokine-activated killer cells, melphalan, mesna, methotrexate,mitoxantrone hydrochloride, monoclonal antibody CD19 (such astisagenlecleucel-T, CART-19, CTL-019), monoclonal antibody CD20,motexafin gadolinium, mycophenolate mofetil, nelarabine, oblimersen,octreotide acetate, omega-3 fatty acids, oxaliplatin, paclitaxel,pegfilgrastim, PEGylated liposomal doxorubicin hydrochloride,pentostatin, perifosine, prednisone, recombinant flt3 ligand,recombinant human thrombopoietin, recombinant interferon alfa,recombinant interleukin-11, recombinant interleukin-12, rituximab,sargramostim, sildenafil citrate (VIAGRA®), simvastatin, sirolimus,tacrolimus, tanespimycin, thalidomide, therapeutic allogeneiclymphocytes, thiotepa, tipifarnib, tositumomab, veltuzumab, vincristinesulfate, vinorelbine ditartrate, vorinostat, WT1 126-134 peptidevaccine, WT-1 analog peptide vaccine, yttrium-90 ibritumomab tiuxetan,yttrium-90 humanized epratuzumab, and any combination thereof.

Examples of therapeutic procedures used to treat WM include peripheralblood stem cell transplantation, autologous hematopoietic stem celltransplantation, autologous bone marrow transplantation, antibodytherapy, biological therapy, enzyme inhibitor therapy, total bodyirradiation, infusion of stem cells, bone marrow ablation with stem cellsupport, in vitro-treated peripheral blood stem cell transplantation,umbilical cord blood transplantation, immunoenzyme techniques, low-LETcobalt-60 gamma ray therapy, bleomycin, conventional surgery, radiationtherapy, and nonmyeloablative allogeneic hematopoietic stem celltransplantation.

Diffuse Large B-cell Lymphoma Combination Therapy

Therapeutic agents used to treat diffuse large B-cell lymphoma (DLBCL)include cyclophosphamide, doxorubicin, vincristine, prednisone,anti-CD20 monoclonal antibodies, etoposide, bleomycin, many of theagents listed for WM, and any combination thereof, such as ICE and RICE.

Chronic Lymphocytic Leukemia Combination Therapy

Examples of therapeutic agents used to treat chronic lymphocyticleukemia (CLL) include chlorambucil, cyclophosphamide, fludarabine,pentostatin, cladribine, doxorubicin, vincristine, prednisone,prednisolone, alemtuzumab, many of the agents listed for WM, andcombination chemotherapy and chemoimmunotherapy, including the followingcommon combination regimens: CVP, R-CVP, ICE, R-ICE, FCR, and FR.

Myelofibrosis Combination Therapy

Myelofibrosis inhibiting agents include, but are not limited to,hedgehog inhibitors, histone deacetylase (HDAC) inhibitors, and tyrosinekinase inhibitors. Non-limiting examples of hedgehog inhibitors aresaridegib and vismodegib.

Examples of HDAC inhibitors include, but are not limited to, pracinostatand panobinostat.

Non-limiting examples of tyrosine kinase inhibitors are lestaurtinib,bosutinib, imatinib, gilteritinib, radotinib, and cabozantinib.

Hyperproliferative Disorder Combination Therapy

Gemcitabine, nab-paclitaxel, and gemcitabine/nab-paclitaxel may be usedwith a CD73 inhibitor and/or TGFβ inhibitor to treat hyperproliferativedisorders.

Bladder Cancer Combination Therapy

Therapeutic agents used to treat bladder cancer include atezolizumab,carboplatin, cisplatin, docetaxel, doxorubicin, fluorouracil (5-FU),gemcitabine, idosfamide, Interferon alfa-2b, methotrexate, mitomycin,nab-paclitaxel, paclitaxel, pemetrexed, thiotepa, vinblastine, and anycombination thereof.

Breast Cancer Combination Therapy

Therapeutic agents used to treat breast cancer include albumin-boundpaclitaxel, anastrozole, capecitabine, carboplatin, cisplatin,cyclophosphamide, docetaxel, doxorubicin, epirubicin, everolimus,exemestane, fluorouracil, fulvestrant, gemcitabine, Ixabepilone,lapatinib, Letrozole, methotrexate, mitoxantrone, paclitaxel, pegylatedliposomal doxorubicin, pertuzumab, tamoxifen, toremifene, trastuzumab,vinorelbine, and any combinations thereof.

Triple Negative Breast Cancer Combination Therapy

Therapeutic agents used to treat triple negative breast cancer includecyclophosphamide, docetaxel, doxorubicin, epirubicin, fluorouracil,paclitaxel, and combinations thereof.

Colorectal Cancer Combination Therapy

Therapeutic agents used to treat colorectal cancer include bevacizumab,capecitabine, cetuximab, fluorouracil, irinotecan, leucovorin,oxaliplatin, panitumumab, ziv-aflibercept, and any combinations thereof.

Castration-Resistant Prostate Cancer Combination Therapy

Therapeutic agents used to treat castration-resistant prostate cancerinclude abiraterone, cabazitaxel, docetaxel, enzalutamide, prednisone,sipuleucel-T, and any combinations thereof.

Esophageal and Esophagogastric Junction Cancer Combination Therapy

Therapeutic agents used to treat esophageal and esophagogastric junctioncancer include capecitabine, carboplatin, cisplatin, docetaxel,epirubicin, fluoropyrimidine, fluorouracil, irinotecan, leucovorin,oxaliplatin, paclitaxel, ramucirumab, trastuzumab, and any combinationsthereof.

Gastric Cancer Combination Therapy

Therapeutic agents used to treat gastric cancer include capecitabine,carboplatin, cisplatin, docetaxel, epirubicin, fluoropyrimidine,fluorouracil, Irinotecan, leucovorin, mitomycin, oxaliplatin,paclitaxel, ramucirumab, trastuzumab, and any combinations thereof.

Head & Neck Cancer Combination Therapy

Therapeutic agents used to treat head & neck cancer include afatinib,bleomycin, capecitabine, carboplatin, cetuximab, cisplatin, docetaxel,fluorouracil, gemcitabine, hydroxyurea, methotrexate, nivolumab,paclitaxel, pembrolizumab, vinorelbine, and any combinations thereof.

Hepatobiliary Cancer Combination Therapy

Therapeutic agents used to treat hepatobiliary cancer includecapecitabine, cisplatin, fluoropyrimidine, 5-fluorourcil, gemecitabine,oxaliplatin, sorafenib, and any combinations thereof.

Hepatocellular Carcinoma Combination Therapy

Therapeutic agents used to treat hepatocellular carcinoma includecapecitabine, doxorubicin, gemcitabine, sorafenib, and any combinationsthereof.

Non-Small Cell Lung Cancer Combination Therapy

Therapeutic agents used to treat non-small cell lung cancer (NSCLC)include afatinib, albumin-bound paclitaxel, alectinib, bevacizumab,bevacizumab, cabozantinib, carboplatin, cisplatin, crizotinib,dabrafenib, docetaxel, erlotinib, etoposide, gemcitabine, nivolumab,paclitaxel, pembrolizumab, pemetrexed, ramucirumab, trametinib,trastuzumab, vandetanib, vemurafenib, vinblastine, vinorelbine, and anycombinations thereof.

Small Cell Lung Cancer Combination Therapy

Therapeutic agents used to treat small cell lung cancer (SCLC) includebendamustime, carboplatin, cisplatin, cyclophosphamide, docetaxel,doxorubicin, etoposide, gemcitabine, ipillimumab, irinotecan, nivolumab,paclitaxel, temozolomide, topotecan, vincristine, vinorelbine, and anycombinations thereof.

Melanoma Combination Therapy

Therapeutic agents used to treat melanoma cancer include albumin boundpaclitaxel, carboplatin, cisplatin, cobiemtinib, dabrafenib,dacrabazine, IL-2, imatinib, interferon alfa-2b, ipilimumab,nitrosourea, nivolumab, paclitaxel, pembrolizumab, pilimumab,temozolomide, trametinib, vemurafenib, vinblastine, and any combinationsthereof.

Ovarian Cancer Combination Therapy

Therapeutic agents used to treat ovarian cancer include 5-flourouracil,albumin bound paclitaxel, altretamine, anastrozole, bevacizumab,capecitabine, carboplatin, cisplatin, cyclophosphamide, docetaxel,doxorubicin, etoposide, exemestane, gemcibabine, ifosfamide, irinotecan,letrozole, leuprolide acetate, liposomal doxorubicin, megestrol acetate,melphalan, olaparib, oxaliplatin, paclitaxel, Pazopanib, pemetrexed,tamoxifen, topotecan, vinorelbine, and any combinations thereof.

Pancreatic Cancer Combination Therapy

Therapeutic agents used to treat pancreatic cancer include5-fluorourcil, albumin-bound paclitaxel, capecitabine, cisplatin,docetaxel, erlotinib, fluoropyrimidine, gemcitabine, irinotecan,leucovorin, oxaliplatin, paclitaxel, and any combinations thereof.

Renal Cell Carcinoma Combination Therapy

Therapeutic agents used to treat renal cell carcinoma include axitinib,bevacizumab, cabozantinib, erlotinib, everolimus, levantinib, nivolumab,pazopanib, sorafenib, sunitinib, temsirolimus, and any combinationsthereof.

5.6 Polynucleotides, Vectors and Methods of Producing Anti-CD73Antibodies

In another aspect, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody described herein or a fragmentthereof (e.g., a light chain variable region and/or heavy chain variableregion) that specifically binds to a CD73 (e.g., human, mouse, orcynomolgus CD73) antigen, and vectors, e.g., vectors comprising suchpolynucleotides for recombinant expression in host cells (e.g., E. coliand mammalian cells). Provided herein are polynucleotides comprisingnucleotide sequences encoding a polypeptide (e.g., a heavy and/or lightchain) of any of the antibodies provided herein, as well as vectorscomprising such polynucleotide sequences, e.g., expression vectors fortheir efficient expression in host cells, e.g., mammalian cells.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular lessthan about 10%) of other material, e.g., cellular material, culturemedium, other nucleic acid molecules, chemical precursors and/or otherchemicals. In a specific embodiment, a nucleic acid molecule(s) encodingan antibody described herein is isolated or purified.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies, which specifically bind to aCD73 (e.g., human, mouse, or cynomolgus CD73) polypeptide and comprisesan amino acid sequence as described herein, as well as antibodies whichcompete with such antibodies for binding to a CD73 (e.g., human, mouse,or cynomolgus CD73) polypeptide (e.g., in a dose-dependent manner), orwhich binds to the same epitope as that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding a polypeptide (e.g., the light chain orheavy chain) of an antibody described herein. The polynucleotides cancomprise nucleotide sequences encoding a light chain comprising the VLFRs and CDRs of antibodies described herein (see, e.g., Table 1) ornucleotide sequences encoding a heavy chain comprising the VH FRs andCDRs of antibodies described herein (see, e.g., Table 1).

Also provided herein are polynucleotides encoding an anti-CD73 (e.g.,human, mouse, or cynomolgus CD73) antibody that are optimized, e.g., bycodon/RNA optimization, replacement with heterologous signal sequences,and elimination of mRNA instability elements. Methods to generateoptimized nucleic acids encoding an anti-CD73 (e.g., human, mouse, orcynomolgus CD73) antibody or a fragment thereof (e.g., light chain,heavy chain, VH domain, or VL domain, optionally linked to a ligandbinding moiety) for recombinant expression by introducing codon changesand/or eliminating inhibitory regions in the mRNA can be carried out byadapting the optimization methods described in, e.g., U.S. Pat. Nos.5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly,all of which are herein incorporated by reference in their entireties.For example, potential splice sites and instability elements (e.g., A/Tor A/U rich elements) within the RNA can be mutated without altering theamino acids encoded by the nucleic acid sequences to increase stabilityof the RNA for recombinant expression. The alterations utilize thedegeneracy of the genetic code, e.g., using an alternative codon for anidentical amino acid. In certain embodiments, it can be desirable toalter one or more codons to encode a conservative mutation, e.g., asimilar amino acid with similar chemical structure and properties and/orfunction as the original amino acid. Such methods can increaseexpression of an anti-CD73 (e.g., human, mouse, or cynomolgus CD73)antibody or fragment thereof by at least 1 fold, 2 fold, 3 fold, 4 fold,5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold,80 fold, 90 fold, or 100 fold or more relative to the expression of ananti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody encoded bypolynucleotides that have not been optimized.

In certain embodiments, an optimized polynucleotide sequence encoding ananti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody describedherein or a fragment thereof (e.g., VL domain and/or VH domain) canhybridize to an antisense (e.g., complementary) polynucleotide of anunoptimized polynucleotide sequence encoding an anti-CD73 (e.g., human,mouse, or cynomolgus CD73) antibody described herein or a fragmentthereof (e.g., VL domain and/or VH domain). In specific embodiments, anoptimized nucleotide sequence encoding an anti-CD73 (e.g., human, mouse,or cynomolgus CD73) antibody described herein or a fragment hybridizesunder high stringency conditions to antisense polynucleotide of anunoptimized polynucleotide sequence encoding an anti-CD73 (e.g., human,mouse, or cynomolgus CD73) antibody described herein or a fragmentthereof. In a specific embodiment, an optimized nucleotide sequenceencoding an anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibodydescribed herein or a fragment thereof hybridizes under high stringency,intermediate or lower stringency hybridization conditions to anantisense polynucleotide of an unoptimized nucleotide sequence encodingan anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody describedherein or a fragment thereof. Information regarding hybridizationconditions has been described, see, e.g., U.S. Patent ApplicationPublication No. US 2005/0048549 (e.g., paragraphs 72-73), which isherein incorporated by reference in its entirety.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies described herein, e.g., antibodiesdescribed in Table 1, and modified versions of these antibodies can bedetermined using methods well known in the art, i.e., nucleotide codonsknown to encode particular amino acids are assembled in such a way togenerate a nucleic acid that encodes the antibody. Such a polynucleotideencoding the antibody can be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier G et al., (1994),BioTechniques 17: 242-6, herein incorporated by reference in itsentirety), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain and/or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody described herein) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR can then be cloned into replicable cloning vectorsusing any method well known in the art.

DNA encoding anti-CD73 (e.g., human, mouse, or cynomolgus CD73)antibodies described herein can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the anti-CD73 (e.g., human, mouse, or cynomolgus CD73)antibodies). Hybridoma cells can serve as a source of such DNA. Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GSSystem™ (Lonza)), or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of anti-CD73 (e.g.,human, mouse, or cynomolgus CD73) antibodies in the recombinant hostcells.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 1 or human gamma 4constant region, and the PCR amplified VL domains can be cloned intovectors expressing a light chain constant region, e.g., human kappa orlambda constant regions. In certain embodiments, the vectors forexpressing the VH or VL domains comprise an EF-1α promoter, a secretionsignal, a cloning site for the variable region, constant domains, and aselection marker such as neomycin. The VH and VL domains can also becloned into one vector expressing the necessary constant regions. Theheavy chain conversion vectors and light chain conversion vectors arethen co-transfected into cell lines to generate stable or transient celllines that express full-length antibodies, e.g., IgG, using techniquesknown to those of skill in the art.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein. In specificembodiments, polynucleotides described herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH domain and/or VL domain provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3, which is herein incorporated by reference in itsentirety.

In certain aspects, provided herein are cells (e.g., host cells)expressing (e.g., recombinantly) antibodies described herein whichspecifically bind to CD73 (e.g., human, mouse, or cynomolgus CD73) andrelated polynucleotides and expression vectors. Provided herein arevectors (e.g., expression vectors) comprising polynucleotides comprisingnucleotide sequences encoding anti-CD73 (e.g., human, mouse, orcynomolgus CD73) antibodies or a fragment for recombinant expression inhost cells, preferably in mammalian cells (e.g., CHO cells). Alsoprovided herein are host cells comprising such vectors for recombinantlyexpressing anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibodiesdescribed herein (e.g., human or humanized antibody). In a particularaspect, provided herein are methods for producing an antibody describedherein, comprising expressing such antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., afull-length antibody, heavy and/or light chain of an antibody, or asingle chain antibody optionally comprising a ligand binding moietydescribed herein) that specifically binds to CD73 (e.g., human, mouse,or cynomolgus CD73) generally involves construction of an expressionvector containing a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody, a polypeptide (e.g., heavy and/orlight chain) of an antibody, or a fragment thereof (e.g., heavy and/orlight chain variable regions) described herein has been obtained, thevector for the production of the antibody can be produced by recombinantDNA technology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody or antibody fragment (e.g., light chain or heavy chain)encoding nucleotide sequence are described herein. Methods which arewell known to those skilled in the art can be used to constructexpression vectors containing antibody or antibody fragment (e.g., lightchain or heavy chain) coding sequences and appropriate transcriptionaland translational control signals. These methods include, for example,in vitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Also provided are replicable vectors comprising anucleotide sequence encoding an antibody described herein, a polypeptide(e.g., heavy or light chain) of an antibody, a heavy or light chainvariable region of an antibody or a fragment thereof, or a heavy orlight chain CDR, operably linked to a promoter. Such vectors can, forexample, include the nucleotide sequence encoding the constant region ofthe antibody molecule (see, e.g., International Publication Nos. WO86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464, which are hereinincorporated by reference in their entireties) and variable regions ofthe antibody can be cloned into such a vector for expression of anentire polypeptide (e.g., the entire heavy chain or the entire lightchain), or both the entire heavy and light chains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody described herein or afragment thereof. Thus, provided herein are host cells containing apolynucleotide encoding an antibody described herein or fragmentsthereof, or a heavy or light chain thereof, or fragment thereof, or asingle chain antibody described herein, operably linked to a promoterfor expression of such sequences in the host cell. In certainembodiments, for the expression of double-chained antibodies, vectorsencoding both the heavy and light chains, individually, can beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below. In certain embodiments, ahost cell contains a vector comprising a polynucleotide encoding boththe heavy chain and light chain of an antibody described herein, or afragment thereof. In specific embodiments, a host cell contains twodifferent vectors, a first vector comprising a polynucleotide encoding aheavy chain or a heavy chain variable region of an antibody describedherein, or a fragment thereof, and a second vector comprising apolynucleotide encoding a light chain or a light chain variable regionof an antibody described herein, or a fragment thereof. In otherembodiments, a first host cell comprises a first vector comprising apolynucleotide encoding a heavy chain or a heavy chain variable regionof an antibody described herein, or a fragment thereof, and a secondhost cell comprises a second vector comprising a polynucleotide encodinga light chain or a light chain variable region of an antibody describedherein. In specific embodiments, a heavy chain/heavy chain variableregion expressed by a first cell associated with a light chain/lightchain variable region of a second cell to form an anti-CD73 (e.g.,human, mouse, or cynomolgus CD73) antibody described herein. In certainembodiments, provided herein is a population of host cells comprisingsuch first host cell and such second host cell.

In a particular embodiment, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-CD73 (e.g., human, mouse,or cynomolgus CD73) antibody described herein, and a second vectorcomprising a polynucleotide encoding a heavy chain/heavy chain variableregion of an anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibodydescribed herein.

A variety of host-expression vector systems can be utilized to expressantibody described herein (see, e.g., U.S. Pat. No. 5,807,715, which isherein incorporated by reference in its entirety). Such host-expressionsystems represent vehicles by which the coding sequences of interest canbe produced and subsequently purified, but also represent cells whichcan, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule described herein in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli and B. subtilis) transformed with, e.g., recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining antibody coding sequences; yeast (e.g., Saccharomyces Pichia)transformed with, e.g., recombinant yeast expression vectors containingantibody coding sequences; insect cell systems infected with, e.g.,recombinant virus expression vectors (e.g., baculovirus) containingantibody coding sequences; plant cell systems (e.g., green algae such asChlamydomonas reinhardtii) infected with, e.g., recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with, e.g., recombinant plasmid expressionvectors (e.g., Ti plasmid) containing antibody coding sequences; ormammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK,HEK-293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3,HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 and BMT10cells) harboring, e.g., recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). In aspecific embodiment, cells for expressing antibodies described hereinare Chinese hamster ovary (CHO) cells, for example CHO cells from theCHO GS System™ (Lonza). In certain embodiments, a polypeptide (e.g., theheavy chain and/or light chain) of an antibody produced by a CHO cellmay have an N-terminal glutamine or glutamate residue replaced bypyroglutamate. In a particular embodiment, cells for expressingantibodies described herein are human cells, e.g., human cell lines. Ina specific embodiment, a mammalian expression vector is pOptiVEC™ orpcDNA3.3. In a particular embodiment, bacterial cells such asEscherichia coli, or eukaryotic cells (e.g., mammalian cells),especially for the expression of whole recombinant antibody, are usedfor the expression of a recombinant antibody. For example, mammaliancells such as CHO cells, in conjunction with a vector such as the majorintermediate early gene promoter element from human cytomegalovirus isan effective expression system for antibodies (Foecking M K & HofstetterH (1986) Gene 45: 101-5; and Cockett M I et al., (1990) Biotechnology8(7): 662-7, each of which is herein incorporated by reference in itsentirety). In certain embodiments, antibodies described herein areproduced by CHO cells or NS0 cells. In a specific embodiment, theexpression of nucleotide sequences encoding antibodies described hereinwhich specifically bind to CD73 (e.g., human, mouse, or cynomolgus CD73)is regulated by a constitutive promoter, inducible promoter or tissuespecific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such an antibodyis to be produced, for the generation of pharmaceutical compositions ofan antibody, vectors which direct the expression of high levels offusion protein products that are readily purified can be desirable. Suchvectors include, but are not limited to, the E. coli expression vectorpUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), in whichthe antibody coding sequence can be ligated individually into the vectorin frame with the lac Z coding region so that a fusion protein isproduced; pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster S M (1989) J Biol Chem 24: 5503-5509);and the like, all of which are herein incorporated by reference in theirentireties. For example, pGEX vectors can also be used to expressforeign polypeptides as fusion proteins with glutathione 5-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to matrixglutathione agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV), for example, can be used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The antibody codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody in infected hosts (e.g., see Logan J & Shenk T(1984) PNAS 81(12): 3655-9, which is herein incorporated by reference inits entirety). Specific initiation signals can also be required forefficient translation of inserted antibody coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression can be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (see,e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, which isherein incorporated by reference in its entirety).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK,HEK-293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (amurine myeloma cell line that does not endogenously produce anyimmunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO,HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20,BMT10 and HsS78Bst cells. In certain embodiments, anti-CD73 (e.g.,human, mouse, or cynomolgus CD73) antibodies described herein areproduced in mammalian cells, such as CHO cells.

In a specific embodiment, the antibodies described herein have reducedfucose content or no fucose content. Such antibodies can be producedusing techniques known one skilled in the art. For example, theantibodies can be expressed in cells deficient or lacking the ability ofto fucosylate. In a specific example, cell lines with a knockout of bothalleles of α1,6-fucosyltransferase can be used to produce antibodieswith reduced fucose content. The Potelligent® system (Lonza) is anexample of such a system that can be used to produce antibodies withreduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an anti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibodydescribed herein can be engineered. In specific embodiments, a cellprovided herein stably expresses a light chain/light chain variableregion and a heavy chain/heavy chain variable region which associate toform an antibody described herein, optionally wherein at least one ofthe light chain/light chain variable region and the heavy chain/heavychain variable region is linked to a ligand binding moiety.

In certain aspects, rather than using expression vectors which containviral origins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA/polynucleotide, engineered cells can be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express an anti-CD73 (e.g., human,mouse, or cynomolgus CD73) antibody described herein or a fragmentthereof Such engineered cell lines can be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (SzybalskaE H & Szybalski W (1962) PNAS 48(12): 2026-2034) and adeninephosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23)genes in tk−, hgprt− or aprt-cells, respectively, all of which areherein incorporated by reference in their entireties. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (WiglerM et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance tothe aminoglycoside G-418 (Wu G Y & Wu C H (1991) Biotherapy 3: 87-95;Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C(1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) AnnRev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) TrendsBiotechnol 11(5): 211-5); and hygro, which confers resistance tohygromycin (Santerre R F et al., (1984) Gene 30(1-3): 147-56), all ofwhich are herein incorporated by reference in their entireties. Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone and such methods aredescribed, for example, in Ausubel F M et al., (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler M, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli N C et al., (eds.), CurrentProtocols in Human Genetics, John Wiley & Sons, N Y (1994);Colbère-Garapin F et al., (1981) J Mol Biol 150: 1-14, all of which areherein incorporated by reference in their entireties.

The expression levels of an antibody can be increased by vectoramplification (for a review, see Bebbington C R & Hentschel C C G, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987), which is herein incorporated by reference in its entirety).When a marker in the vector system expressing antibody is amplifiable,increase in the level of inhibitor present in culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse G F et al., (1983) Mol Cell Biol 3: 257-66,which is herein incorporated by reference in its entirety).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain derivedpolypeptides. The host cells can be co-transfected with differentamounts of the two or more expression vectors. For example, host cellscan be transfected with any one of the following ratios of a firstexpression vector and a second expression vector: about 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35,1:40, 1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot N J (1986) Nature322: 562-565; and Kohler G (1980) PNAS 77: 2197-2199, each of which isherein incorporated by reference in its entirety). The coding sequencesfor the heavy and light chains can comprise cDNA or genomic DNA. Theexpression vector can be monocistronic or multicistronic. Amulticistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9,10 or more genes/nucleotide sequences, or in the range of 2-5, 5-10, or10-20 genes/nucleotide sequences. For example, a bicistronic nucleicacid construct can comprise, in the following order, a promoter, a firstgene (e.g., heavy chain of an antibody optionally linked to a ligandbinding moiety as described herein), and a second gene and (e.g., lightchain of an antibody optionally linked to a ligand binding moiety asdescribed herein). In such an expression vector, the transcription ofboth genes can be driven by the promoter, whereas the translation of themRNA from the first gene can be by a cap-dependent scanning mechanismand the translation of the mRNA from the second gene can be by acap-independent mechanism, e.g., by an IRES.

Once an antibody described herein has been produced by recombinantexpression, it can be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an antibody described herein is isolated orpurified. Generally, an isolated antibody is one that is substantiallyfree of other antibodies with different antigenic specificities than theisolated antibody. For example, in a particular embodiment, apreparation of an antibody described herein is substantially free ofcellular material and/or chemical precursors. The language“substantially free of cellular material” includes preparations of anantibody in which the antibody is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, anantibody that is substantially free of cellular material includespreparations of antibody having less than about 30%, 20%, 10%, 5%, 2%,1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referredto herein as a “contaminating protein”) and/or variants of an antibody,for example, different post-translational modified forms of an antibodyor other different versions of an antibody (e.g., antibody fragments).When the antibody is recombinantly produced, it is also generallysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of theprotein preparation. When the antibody is produced by chemicalsynthesis, it is generally substantially free of chemical precursors orother chemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the antibody have less than about 30%,20%, 10%, or 5% (by dry weight) of chemical precursors or compoundsother than the antibody of interest. In a specific embodiment,antibodies described herein are isolated or purified.

Antibodies or fragments thereof that specifically bind to CD73 (e.g.,human, mouse, or cynomolgus CD73) can be produced by any method known inthe art for the synthesis of antibodies, for example, by chemicalsynthesis or by recombinant expression techniques. The methods describedherein employ, unless otherwise indicated, conventional techniques inmolecular biology, microbiology, genetic analysis, recombinant DNA,organic chemistry, biochemistry, PCR, oligonucleotide synthesis andmodification, nucleic acid hybridization, and related fields within theskill of the art. These techniques are described, for example, in thereferences cited herein and are fully explained in the literature. See,e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989),Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY; Ausubel F M et al., Current Protocols in Molecular Biology,John Wiley & Sons (1987 and annual updates); Current Protocols inImmunology, John Wiley & Sons (1987 and annual updates) Gait (ed.)(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press;Eckstein (ed.) (1991) Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: ALaboratory Manual, Cold Spring Harbor Laboratory Press, all of which areherein incorporated by reference in their entireties.

In a specific embodiment, an antibody described herein is an antibody(e.g., recombinant antibody) prepared, expressed, created or isolated byany means that involves creation, e.g., via synthesis, geneticengineering of DNA sequences. In certain embodiments, such an antibodycomprises sequences (e.g., DNA sequences or amino acid sequences) thatdo not naturally exist within the antibody germline repertoire of ananimal or mammal (e.g., human) in vivo.

In one aspect, provided herein is a method of making an antibody whichspecifically bind to CD73 (e.g., human, mouse, or cynomolgus CD73)comprising culturing a cell or host cell described herein. In oneembodiment, the method is performed in vitro. In a certain aspect,provided herein is a method of making an antibody which specificallybind to CD73 (e.g., human, mouse, or cynomolgus CD73) comprisingexpressing (e.g., recombinantly expressing) the antibody using a cell orhost cell described herein (e.g., a cell or a host cell comprisingpolynucleotides encoding an antibody described herein). In a particularembodiment, the cell is an isolated cell. In a particular embodiment,the exogenous polynucleotides have been introduced into the cell. In aparticular embodiment, the method further comprises the step ofpurifying the antibody obtained from the cell or host cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York, whichis herein incorporated by reference in its entirety).

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow E & Lane D,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which isherein incorporated by reference in its entirety. The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. For example, monoclonal antibodies can be producedrecombinantly from host cells exogenously expressing a polypeptide of anantibody described herein or a fragment thereof, for example, lightchain and/or heavy chain of such antibody.

In specific embodiments, a “monoclonal antibody,” as used herein, is anantibody produced by a single cell (e.g., hybridoma or host cellproducing a recombinant antibody), wherein the antibody specificallybinds to CD73 (e.g., human, mouse, or cynomolgus CD73) as determined,e.g., by ELISA or other antigen-binding or competitive binding assayknown in the art or in the examples provided herein. In particularembodiments, a monoclonal antibody can be a chimeric antibody or ahumanized antibody. In certain embodiments, a monoclonal antibody is amonovalent antibody or multivalent (e.g., bivalent) antibody. Inparticular embodiments, a monoclonal antibody is a monospecific ormultispecific antibody (e.g., bispecific antibody). Monoclonalantibodies described herein can, for example, be made by the hybridomamethod as described in Kohler G & Milstein C (1975) Nature 256: 495,which is herein incorporated by reference in its entirety, or can, e.g.,be isolated from phage libraries using the techniques as describedherein, for example. Other methods for the preparation of clonal celllines and of monoclonal antibodies expressed thereby are well known inthe art (see, for example, Chapter 11 in: Short Protocols in MolecularBiology, (2002) 5th Ed., Ausubel F M et al., supra).

As used herein, an antibody binds to an antigen multivalently (e.g.,bivalently) when the antibody comprises at least two (e.g., two or more)monovalent binding domains, each monovalent binding domain capable ofbinding to an epitope on the antigen. Each monovalent binding domain canbind to the same or different epitopes on the antigen.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. For example,in the hybridoma method, a mouse or other appropriate host animal, suchas a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunizedto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein (e.g., CD73 (e.g.,human, mouse, or cynomolgus CD73)) used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding J W (Ed), MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986),herein incorporated by reference in its entirety). Additionally, a RIMMS(repetitive immunization multiple sites) technique can be used toimmunize an animal (Kilpatrick K E et al., (1997) Hybridoma 16:381-9,herein incorporated by reference in its entirety).

In certain embodiments, mice (or other animals, such as rats, monkeys,donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen(e.g., CD73 (e.g., human, mouse, or cynomolgus CD73)) and once an immuneresponse is detected, e.g., antibodies specific for the antigen aredetected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example, cells from cellline SP20 available from the American Type Culture Collection (ATCC®)(Manassas, VA), to form hybridomas. Hybridomas are selected and clonedby limited dilution. In certain embodiments, lymph nodes of theimmunized mice are harvested and fused with NS0 myeloma cells.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asthe NS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,CA, USA, and SP-2 or X63-Ag8.653 cells available from the American TypeCulture Collection, Rockville, MD, USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor D (1984) J Immunol 133: 3001-5;Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), each ofwhich is herein incorporated by reference in its entirety).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against CD73 (e.g., human,mouse, or cynomolgus CD73). The binding specificity of monoclonalantibodies produced by hybridoma cells is determined by methods known inthe art, for example, immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbentassay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding J W (Ed), Monoclonal Antibodies: Principles and Practice,supra). Suitable culture media for this purpose include, for example,D-MEM or RPMI 1640 medium. In addition, the hybridoma cells may be grownin vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Antibodies described herein include, e.g., antibody fragments whichrecognize a specific CD73 (e.g., human, mouse, or cynomolgus CD73), andwhich can be generated by any technique known to those of skill in theart. For example, Fab and F(ab′)₂ fragments described herein can beproduced by proteolytic cleavage of immunoglobulin molecules, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments). A Fab fragment corresponds to one of the twoidentical arms of an antibody and contains the complete light chainpaired with the VH and CH1 domains of the heavy chain. A F(ab′)₂fragment contains the two antigen-binding arms of an antibody moleculelinked by disulfide bonds in the hinge region.

Further, the antibodies described herein can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, DNA sequences encoding VH and VL domains are amplifiedfrom animal cDNA libraries (e.g., human or murine cDNA libraries ofaffected tissues). The DNA encoding the VH and VL domains are recombinedtogether with a scFv linker by PCR and cloned into a phagemid vector.The vector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13, and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to a particular antigencan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Examples ofphage display methods that can be used to make the antibodies describedherein include those disclosed in Brinkman U et al., (1995) J ImmunolMethods 182: 41-50; Ames R S et al., (1995) J Immunol Methods 184:177-186; Kettleborough C A et al., (1994) Eur J Immunol 24: 952-958;Persic L et al., (1997) Gene 187: 9-18; Burton D R & Barbas C F (1994)Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134;International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO 97/13844; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727, 5,733,743 and 5,969,108, all of which are herein incorporatedby reference in their entireties.

In addition, the antibodies described herein can also be generated usingspecific immunization methods, alone or in a combination with phagedisplay technologies as known in the art. For example, DNA immunizationhas been recently reported as an efficient strategy for vaccination. SeeBolhassani and Yazdi (2009) Avicenna J Med Biotechnol. 1:71-88, Liu etal. (2016) Emerg Microbes Infect. 5:e33, and van der Woning et al.(2016) MAbs. 8:1126-1135, each of which is herein incorporated byreference in its entirety. Immunizing animals with DNA encoding aprotein of interest can be performed using the similar methods astraditional animal immunization using the protein itself, and caninclude similar initial immunization and boosting steps. After DNAimmunization, hybridoma cells can be prepared. Alternatively oradditionally, nucleic acid molecules (e.g., RNAs) can be isolated fromtissues (e.g., spleen) of the immunized animal to generate a phasedisplay library for antibody panning and screening.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceantibody fragments such as Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax R L et al., (1992) BioTechniques12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; andBetter M et al., (1988) Science 240: 1041-1043, all of which are hereinincorporated by reference in their entireties.

In certain embodiments, to generate whole antibodies, PCR primersincluding VH or VL nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site can be used to amplifythe VH or VL sequences from a template, e.g., scFv clones. Utilizingcloning techniques known to those of skill in the art, the PCR amplifiedVH domains can be cloned into vectors expressing a VH constant region,and the PCR amplified VL domains can be cloned into vectors expressing aVL constant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415, all of which are herein incorporated byreference in their entireties.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₂, IgG₃ and IgG₄. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 andEP 519596; Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G Met al., (1994) Prot Engineering 7(6): 805-814; and Roguska M A et al.,(1994) PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,International Publication No. WO 93/17105; Tan P et al., (2002) JImmunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al.,(1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996) ProteinEng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23 Supp):5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22; SandhuJ S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) J MolBiol 235(3): 959-73, all of which are herein incorporated by referencein their entireties. See also U.S. Application Publication No. US2005/0042664 A1 (Feb. 24, 2005), which is herein incorporated byreference in its entirety.

Methods for making multispecific (e.g., bispecific antibodies) have beendescribed, see, for example, U.S. Pat. Nos. 7,951,917; 7,183,076;8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713, allof which are herein incorporated by reference in their entireties.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well known in the art. See RiechmannL & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al.,(2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) JBiotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591 and WO 01/44301, all of whichare herein incorporated by reference in their entireties.

Further, antibodies that specifically bind to a CD73 (e.g., human,mouse, or cynomolgus CD73) antigen can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” an antigen using techniques wellknown to those skilled in the art. See, e.g., Greenspan N S & Bona C A(1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8):2429-2438, each of which is herein incorporated by reference in itsentirety.

In particular embodiments, an antibody described herein, which binds tothe same epitope of CD73 (e.g., human, mouse, or cynomolgus CD73) as ananti-CD73 (e.g., human, mouse, or cynomolgus CD73) antibody describedherein, is a human antibody. In particular embodiments, an antibodydescribed herein, which competitively blocks (e.g., in a dose-dependentmanner) any one of the antibodies described herein, from binding to CD73(e.g., human, mouse, or cynomolgus CD73), is a human antibody. Humanantibodies can be produced using any method known in the art. Forexample, transgenic mice which are incapable of expressing functionalendogenous immunoglobulins, but which can express human immunoglobulingenes, can be used. In particular, the human heavy and light chainimmunoglobulin gene complexes can be introduced randomly or byhomologous recombination into mouse embryonic stem cells. Alternatively,the human variable region, constant region, and diversity region can beintroduced into mouse embryonic stem cells in addition to the humanheavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of an antigen (e.g., CD73 (e.g., human, mouse, or cynomolgusCD73)). Monoclonal antibodies directed against the antigen can beobtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg N & Huszar D (1995) Int RevImmunol 13:65-93, herein incorporated by reference in its entirety. Fora detailed discussion of this technology for producing human antibodiesand human monoclonal antibodies and protocols for producing suchantibodies, see, e.g., International Publication Nos. WO 98/24893, WO96/34096 and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126,5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598, allof which are herein incorporated by reference in their entireties.Examples of mice capable of producing human antibodies include theXenomouse™ (Abgenix, Inc.; U.S. Pat. Nos. 6,075,181 and 6,150,184), theHuAb-Mouse™ (Mederex, Inc./Gen Pharm; U.S. Pat. Nos. 5,545,806 and5,569,825), the Trans Chromo Mouse™ (Kirin) and the KM Mouse™(Medarex/Kirin), all of which are herein incorporated by reference intheir entireties.

Human antibodies that specifically bind to CD73 (e.g., human, mouse, orcynomolgus CD73) can be made by a variety of methods known in the artincluding the phage display methods described above using antibodylibraries derived from human immunoglobulin sequences. See also U.S.Pat. Nos. 4,444,887, 4,716,111, and 5,885,793; and InternationalPublication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741, all of which are hereinincorporated by reference in their entireties.

In certain embodiments, human antibodies can be produced usingmouse-human hybridomas. For example, human peripheral blood lymphocytestransformed with Epstein-Barr virus (EBV) can be fused with mousemyeloma cells to produce mouse-human hybridomas secreting humanmonoclonal antibodies, and these mouse-human hybridomas can be screenedto determine ones which secrete human monoclonal antibodies thatspecifically bind to a target antigen (e.g., CD73 (e.g., human, mouse,or cynomolgus CD73)). Such methods are known and are described in theart, see, e.g., Shinmoto H et al., (2004) Cytotechnology 46: 19-23;Naganawa Y et al., (2005) Human Antibodies 14: 27-31, each of which isherein incorporated by reference in its entirety.

5.7 Kits

Also provided are kits comprising one or more antibodies describedherein, or pharmaceutical compositions or conjugates thereof. In aspecific embodiment, provided herein is a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions described herein, such asone or more antibodies provided herein. In certain embodiments, the kitscontain a pharmaceutical composition described herein and anyprophylactic or therapeutic agent, such as those described herein. Incertain embodiments, the kits may contain a T cell mitogen, such as,e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA),or a TCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody. Optionally associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

Also provided, are kits that can be used in the above methods. In oneembodiment, a kit comprises an antibody described herein, preferably apurified antibody, in one or more containers. In a specific embodiment,kits described herein contain a substantially isolated CD73 (e.g.,human, mouse, or cynomolgus CD73) antigen as a control. In anotherspecific embodiment, the kits described herein further comprise acontrol antibody which does not react with a CD73 (e.g., human, mouse,or cynomolgus CD73) antigen. In another specific embodiment, kitsdescribed herein contain one or more elements for detecting the bindingof an antibody to a CD73 (e.g., human, mouse, or cynomolgus CD73)antigen (e.g., the antibody can be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody can be conjugated to a detectablesubstrate). In specific embodiments, a kit provided herein can include arecombinantly produced or chemically synthesized CD73 (e.g., human,mouse, or cynomolgus CD73) antigen. The CD73 (e.g., human, mouse, orcynomolgus CD73) antigen provided in the kit can also be attached to asolid support. In a more specific embodiment, the detecting means of theabove described kit includes a solid support to which a CD73 (e.g.,human, mouse, or cynomolgus CD73) antigen is attached. Such a kit canalso include a non-attached reporter-labeled anti-human antibody oranti-mouse/rat antibody. In this embodiment, binding of the antibody tothe CD73 (e.g., human, mouse, or cynomolgus CD73) antigen can bedetected by binding of the said reporter-labeled antibody. In oneembodiment, the present invention relates to the use of a kit of thepresent invention for in vitro assaying and/or detecting CD73 antigen(e.g., human, mouse, or cynomolgus CD73) in a biological sample.

6. EXAMPLES

The examples in this Section (i.e., Section 6) are offered by way ofillustration, and not by way of limitation.

6.1 Example 1: Characterization of an Anti-CD73 Antibody

This example describes the generation and characterization of antibodiesthat bind to CD73 (e.g., human CD73, cynomolgus CD73, or mouse CD73). Inparticular, this example describes murine and chimeric antibodies thatspecifically bind to human CD73, inhibit the enzymatic activity of humanCD73, and mediate human CD73 internalization upon binding.

6.1.1 Generation of Anti-CD73 Antibodies

Anti-CD73 antibodies were identified by immunizing mice with eitherrecombinant human CD73 protein or DNA encoding human CD73, amplifyingnucleic acid sequences (e.g., RNA or complementary DNA sequences)encoding heavy chain variable regions and light chain variable regionsfrom the splenic B cells of the immunized mice, assembling thesesequences into vectors to form a proprietary phage display library, andselecting phage clones that produced antibodies having binding affinityto human CD73 (NCBI Reference Sequence: AAH65937, SEQ ID NO: 129, R&DSystems, 5795EN). Three selected antibodies, designated BA010, BA011,and BA012, were further characterized. The heavy chain variable regionand light chain variable region amino acid sequences of BA010, BA011,and BA012 are set forth in SEQ ID NOs: 19 and 33; 20 and 34; and 21 and35, respectively. Constant regions were fused to these variable regionsto form the antibodies described herein. For example, the murineantibody BA012 comprises an Igκ light chain (SEQ ID NO: 92) and an IgG2aheavy chain having a N297A substitution (SEQ ID NO: 64), numberedaccording to the EU numbering system.

Chimeric antibodies were produced by grafting the variable regions ofthe heavy and light chains of BA010, BA011, and BA012 to the constantregion of human IgG₁ heavy chain having a N297A substitution, numberedaccording to the EU numbering system, and the constant region of humanIgκ light chain, respectively. The resulting chimeric antibodiesoriginating from BA010, BA011, and BA012 are designated BA013, BA014,and BA015, as set forth in SEQ ID NOs: 65 and 94, 67 and 95, and 70 and96, respectively, for their heavy and light chains. Similarly, otherchimeric antibodies were produced by grafting the variable regions ofthe heavy and light chains of BA012 to various human constant regions.For example, BA016, as set forth in SEQ ID NOs: 71 (heavy chain) and 96(light chain), comprises a human IgG₂ constant domain. BA017, as setforth in SEQ ID NOs: 73 (heavy chain) and 96 (light chain), comprises ahuman IgG₁-A330S-P331S/IgG₂ hybrid constant domain. BA018, as set forthin SEQ ID NOs: 75 (heavy chain) and 96 (light chain), comprises a humanIgG₁-N297A/IgG₂ hybrid constant domain.

6.1.2 Binding of Anti-CD73 Antibodies to Recombinant CD73

Surface plasmon resonance (SPR) was used to determine the affinity ofBA013, BA014, and BA015 to recombinant CD73 using the BIAcore® T200system (GE Healthcare). Specifically, 2 μg/ml of antibodies diluted in arunning buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05%surfactant P20) were captured on a Series S Sensor Chip Protein A (GEHealthcare Ltd, cat #29-1275-56) using a 15 sec injection at a flow rateof 10 μl/min to reach about 80 resonance units (RUs). Recombinant human,cynomolgus, or mouse CD73 protein fused with a 6×His-tag (R&D Systems,5795EN; Sino Biological, 90192-C08H; and R&D Systems, 4488EN,respectively) diluted in the running buffer at the concentration ofabout 0.14 to 33 nM were flowed over the chip surface at a flow rate of30 μl/min with a 5-min association phase and a 20-min dissociationphase. The sensor chip was regenerated between cycles with a 30-secinjection of 10 mM glycine, pH 1.5. Sensograms were evaluated and fit toa simple Langmuir 1:1 interaction model using the global data analysisoption of BiaEvaluation 3.1 software. Data quality was verified byvisually inspecting deviations and curve fitting, and by evaluating theparameters of Rmax, Chi2, and Tc. The binding kinetics (K_(a), K_(d) andK_(D)) were determined from the sensorgram analyses and are shown inTables 7 and 8. Two reference anti-CD73 antibodies, named ReferenceAntibody 1 (“RA001”) and Reference Antibody 2 (“RA002”), were used forcomparison.

TABLE 7 SPR-based affinity measurements for anti-CD73 antibodies HumanCD73 Cynomolgus CD73 Mouse CD73 K_(a) K_(d) K_(D) K_(a) K_(d) K_(D)K_(a) K_(d) K_(D) Clone (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) (1/Ms) (1/s)(nM) BA013 1.0E+06 2.9E−03 2.8  3.0E+05 2.0E−04 0.7 4.2E+04 6.1E−04 14.5BA014 4.6E+07 4.2E−04 0.010 5.0E+04 3.0E−05 0.6 2.5E+04 2.3E−04  9.4BA015 2.8E+07 4.1E−04 0.015 3.1E+05 8.7E−05 0.3 6.1E+04 6.0E−04 10  

TABLE 8 Affinity comparison between BA015 and Reference anti-CD73antibodies Human CD73 Cynomolgus CD73 K_(a) K_(d) K_(D) K_(a) K_(d)K_(D) Clone (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) BA015 2.27E+06 2.00E−040.088 1.14E+06 8.30E−05 0.07 RA001 7.73E+05 3.49E−05 0.045 6.81E+052.04E−07 0.03 RA002 1.07E+06 1.99E−04 0.186 4.55E+05 1.52E−05 0.3 

6.1.3 Binding of Anti-CD73 Antibodies to CD73-Expressing Cells

The affinity of the anti-CD73 antibodies BA013, BA014, and BA015 formembrane-associated CD73 was determined by assessing their binding tocells ectopically expressing full-length human, cynomolgus, or mouseCD73. Briefly, Chinese hamster ovarian (CHO) cells were transduced withlentiviral vectors comprising a mouse, cynomolgus, or human CD73 codingsequence under the control of a CMV promoter, and stable CHO-CD73 cellswere generated by antibiotic selection. Expression of CD73 was verifiedby flow cytometry using positive control antibodies. The stable CHO-CD73cells were grown in suspension culture in RPMI media supplemented with10% heat-inactivated FBS at 37′C and 5% CO₂. For binding analysis, thestable CHO-CD73 cells were incubated for 30 minutes at 4° C. with aneight-point dose titration of a test or isotype control antibody from0.005 to 10 μg/ml diluted in FACS buffer (PBS, 2 mM EDTA, 0.5% BSA, pH7.2). RA001, which has an Igλ light chain, was used for comparison. Thecells were washed twice in FACS buffer and incubated withFITC-conjugated mouse anti-human kappa detection antibody (for BA013,BA014, and BA015; Life Technologies, HP6062, 1:100 dilution in FACSbuffer) or FITC-conjugated goat-anti-human lambda detection antibody(for Reference Antibody 1; BD Biosciences, 644139, 1:100 dilution inFACS buffer) for 30 minutes at 4° C. The cells were then washed twiceand analyzed using the LSRFortessa™ flow cytometer (BD Biosciences).FACS data were analyzed using FACS DIVA and WEHI Weasel software, andwere plotted with Graphpad Prism software.

As shown in FIG. 1 , BA013, BA014, BA015, and RA001 each exhibiteddose-dependent binding to mouse, cynomolgus, and human CD73.

6.1.4 Inhibition of CD73 Enzymatic Activity by Anti-CD73 Antibodies

The ability of the anti-CD73 antibodies to inhibit the enzymaticactivity of CD73 was assessed on live cells ectopically expressing humanCD73. Briefly, Jurkat cells or CHO cells were transduced with lentiviralvectors comprising a human CD73 coding sequence under the control of anEF1a promoter. Stable cell lines were generated by fluorescenceactivated cell sorting (FACS ARIA Fusion), and the expression of CD73was verified by flow cytometry using a positive control anti-CD73antibody RA001. Cells were grown in suspension culture in RPMI mediasupplemented with 10% heat-inactivated FBS at 37° C. and 5% CO₂. Forenzymatic activity analysis, the CHO or Jurkat cells were plated inflat-bottom tissue culture plates at a density of 5×10⁴ cells per well.An eight-point dose titration of an anti-CD73 antibody or isotypecontrol antibody from 0.005 to 10 μg/ml was added to the cells to reacha final volume of 50 μl/well. After a one-hour incubation, 25 μl of 300μM adenosine monophosphate (AMP) was added to each well to allowhydrolysis of AMP by CD73 for 20 minutes at 37° C. and 5% CO₂. The cellswere then pelleted, and approximately 75 μl of supernatant was mixedwith 25 μl of 12 μM adenosine triphosphate (ATP) in a white flat-bottomtissue culture plate. 100 μl of reconstituted CellTiter Glo® (Promega)was added to each well immediately afterwards, and luminescence wasrecorded using Envision instrument (Perkin Elmer) after 5 minutes.

The luminescence levels correlate with the CD73 activity in each sample,because AMP inhibits the luciferase reaction, and hydrolysis of AMP byCD73 relieves the inhibition. Thus, as shown in FIG. 2A, BA013, BA014,BA015, and RA001 each inhibited the enzymatic activity of CD73 expressedon Jurkat cells. The calculated area under the curve (AUC) values foranti-CD73 antibodies in FIG. 2A are presented in Table 9. Similarinhibition by BA015 was observed using CD73-expressing CHO cells (FIG.2B).

TABLE 9 AUC values for anti-CD73 antibodies in FIG. 2A Antibody AUCBA013 215.2 BA015 239 BA014 260 RA001 407.9

6.1.5 Internalization of Anti-CD73 Antibodies Upon CD73 Binding

The internalization of anti-CD73 antibodies into cells was analyzedusing a cell toxicity assay, and antibody-mediated internalization ofCD73 was assessed using a live-cell imaging method.

The cell toxicity assay employed an antibody-drug conjugate αHFc-NC-MMAE(anti-human IgG Fc antibody conjugated to monomethyl auristatin E(MMAE), with a non-cleavable linker, Moradec LLC), which could transportthe cytotoxic payload MMAE into the cytoplasm upon internalization.Briefly, a Jurkat cell line ectopically expressing human CD73, asdescribed in Section 6.1.4, was plated in white flat-bottom tissueculture plates at a density of 2×10⁴ per well. An eight-point dosetitration of an anti-CD73 antibody or isotype control antibody from0.005 to 10 μg/ml, along with αHFc-NC-MMAE at the same concentration asthe antibody, was added to the cells to reach a final volume of 100μl/well. Following a 72-hour incubation at 37° C. and 5% CO₂, 90 μl ofreconstituted CellTiter Glo® (Promega) was added to each well, andluminescence, representing cell viability, was recorded using Envisioninstrument (Perkin Elmer) after 5 minutes.

As shown in FIG. 3A, incubation with BA015, RA001, or RA002, along withαHFc-NC-MMAE, caused cell death of CD73-expressing Jurkat cells in adose-dependent manner. The calculated AUC values for anti-CD73antibodies in FIG. 3A are presented in Table 10. This result suggestedthat BA015, RA001, and RA002 were internalized upon CD73 binding.

TABLE 10 AUC values for anti-CD73 antibodies in FIG. 3A Antibody AUCBA015 20.81 RA002 30.92 RA001 35.98

Imaging by confocal fluorescence microscopy of live cells revealedinternalization of CD73. Specifically, Jurkat cells ectopicallyexpressing a HaloTag®-CD73 fusion protein, generated by a similar methodas described in Section 6.1.4, were first incubated with 1 μM CellTraceFar Red Cell Proliferation Dye (Life Technologies) for 30 minutes at 37°C. and 5% CO2 to label the cell bodies. The cells were then washed inPBS and stained with a membrane-impermeable HaloTag® Alexa Fluor™ 488ligand (Promega, 1 μM) for 15 minutes at 37° C. and 5% CO₂ to label theHaloTag®-CD73 fusion protein on the cell surface. The labeled cells wereresuspended in fresh culture media at the density of 15,000 cells perwell in a 384-well microscopy plate, and were incubated with 10 μg/ml ofan anti-CD73 antibody or isotype control antibody. Live images werecollected using an ImageXpress Micro Confocal High-Content microscope(Molecular Devices) at 37° C. and 5% CO2, and images were acquired inthe Cy5 channel (CellTrace Far Red Cell Proliferation Dye) and FITCchannel (HaloTag® Alexa Fluor™ 488) every hour over a course of 6 hours.In total, for each condition at each time point, four images (40×magnification) were acquired resulting in analysis of an average of 648cells (±87 cells, standard deviation). Image analysis to quantify theamount of internalized CD73 was performed using MetaXpress analysissoftware (Molecular Devices).

As shown in FIGS. 3B (representative images) and 3C (quantification ofimaging data), BA015 and RA001 each promoted rapid CD73 internalization,as demonstrated by the fluorescence of Alexa Fluor™ 488 in the cytosol.

6.2 Example 2: Generation and Characterization of Humanized Anti-CD73Antibodies

This example describes the generation and characterization of humanizedantibodies that specifically bind to CD73 (e.g., human CD73, cynomolgusCD73, and mouse CD73).

6.2.1 Generation of Humanized Anti-CD73 Antibodies

Humanized variants of BA012 were generated by grafting the heavy chainand light chain CDR sequences of BA012 into human antibody frameworkregions. An exemplary humanized variant, BA019, comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 77 and a light chaincomprising the amino acid sequence of SEQ ID NO: 97. Variantsincorporated amino acid substitutions G24A/R98S in the heavy chainvariable region (VH), and/or F71Y in the light chain variable region(V_(L)), in the human framework regions. In addition, variantsincorporated amino acid substitutions in the CDRs, such as N55S/N61S orN55A/N61A in CDRH2, and N31I/Y32S in CDRL1. The sequences of thehumanized antibodies BA019-BA034 are disclosed in Tables 1-4. Inparticular, a humanized anti-CD73 antibody designated BA020 comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 79 and alight chain comprising the amino acid sequence of SEQ ID NO: 97. Anotherhumanized anti-CD73 antibody designated BA025 comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 85 and a light chaincomprising the amino acid sequence of SEQ TD NO: 97.

6.2.2 Binding of Humanized and Chimeric Anti-CD73 Antibodies toRecombinant CD73

The affinity of some humanized anti-CD73 antibodies (BA019-BA022 andBA024-BA030) to human or cynomolgus CD73 were determined by SPR usingthe method as described in Section 6.1.2. BA012, RA001, and RA002 wereused as controls. The binding kinetics (K_(a), K_(d) and K_(D))determined from the binding curves are shown in Table 11.

TABLE 11 Affinity of anti-CD73 antibodies to CD73 as measured by SPRHuman CD73 Cynomolgus CD73 K_(a) K_(d) K_(D) K_(a) K_(d) K_(D) Clone(1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) RA001 7.39E+05 1.24E−05 0.023.38E+05 2.96E−05 0.09 RA002 4.14E+05 7.19E−05 0.17 3.57E+05 7.49E−050.21 BA012 1.46E+06 6.17E−05 0.04 1.47E+06 7.23E−05 0.05 BA019 1.21E+061.21E−04 0.1 9.88E+05 1.28E−04 0.13 BA020 1.12E+06 7.35E−05 0.061.06E+06 9.83E−05 0.09 BA021 9.82E+05 1.66E−04 0.17 9.38E+05 1.82E−040.19 BA022 1.07E+06 8.57E−05 0.08 1.01E+06 1.05E−04 0.1 BA024 1.09E+067.43E−04 0.68 9.20E+05 6.45E−04 0.7 BA025 1.22E+06 2.29E−04 0.191.14E+06 2.60E−04 0.23 BA026 1.13E+06 1.85E−04 0.17 1.05E+06 2.16E−040.21 BA027 1.23E+06 9.40E−04 0.76 1.16E+06 9.83E−04 0.85 BA028 1.13E+061.20E−03 1.1 1.06E+06 1.29E−03 1.2 BA029 1.20E+06 4.42E−04 0.37 1.13E+064.70E−04 0.42 BA030 1.08E+06 3.63E−04 0.34 1.02E+06 3.95E−04 0.39

Similarly, the binding kinetics for chimeric (murine variabledomain+human constant domain) antibodies BAO16 and BAO18 are shown inTable 12. Specifically, different Fc variants for RA001 and RA002, aswell as murine antibody BAO12, were used for comparison.

TABLE 12 Affinity comparison between anti-CD73 antibodies with differentFc regions Human CD73 Cynomolgus CD73 Mouse CD73 K_(a) K_(d) K_(D) K_(a)K_(d) K_(D) K_(a) K_(d) K_(D) Clone Fc (1/Ms) (1/s) (nM) (1/Ms) (1/s)(nM) (1/Ms) (1/s) (nM) BA012 IgG1-N297A 1.47E+06 8.11E−05 0.06 1.36E+069.46E−05 0.07 8.90E+06 7.07E−03 0.79 BA016 IgG2 1.33E+06 3.38E−04 0.251.29E+06 3.27E−04 0.25 3.15E+06 7.85E−03 2.50 BA018 IgG1/IgG2 hybrid1.40E+06 1.51E−04 0.10 1.41E+06 1.65E−04 0.01 8.37E+06 1.58E−02 1.90RA001 IgG1-N297A 3.16E+05 1.89E−05 0.06 2.95E+05 1.66E−05 0.06 8.88E+042.19E−05 0.25 IgG2 4.17E+05 8.85E−05 0.21 3.39E+05 1.09E−04 0.321.50E+05 9.95E−05 0.70 IgG1/IgG2 hybrid 3.25E+05 1.30E−05 0.04 3.08E+052.48E−05 0.09 1.40E+05 4.36E−06 0.03 RA002 IgG1-N297A 3.71E+05 7.37E−050.20 3.89E+05 8.19E−05 0.21 NB NB NB IgG4-S228P 4.14E+05 7.19E−05 0.173.57E+05 7.49E−05 0.21 NB NB NB NB: no binding

6.2.3 Binding of Humanized Anti-CD73 Antibodies to CD73-Expressing Cells

The affinities of the humanized anti-CD73 antibodies tomembrane-associated CD73 were determined. Specifically, CHO cellsectopically expressing full-length human or cynomolgus CD73 weregenerated, and the binding of the antibodies to the cells were examined,using the method as described in Example 6.1.3. BA015, RA1, and RA02were also examined for comparison.

As shown in FIGS. 4A-4P and 5A-5P, each of the tested humanizedantibodies, BAO19-BA030, as well as chimeric antibody BAO15 and the tworeference antibodies, but not the isotype control, exhibiteddose-dependent binding to cynomolgus CD73 (FIGS. 4A-4P) and human CD73(FIGS. 5A-5P). The calculated AUC values for anti-CD73 antibodies inFIGS. 4B-4P and FIGS. 5B-5P are presented in Table 13 and Table 14,respectively.

TABLE 13 AUC values for anti-CD73 antibodies in FIGS. 4B-4P Antibody AUCRA001 1848 BA026 1881 BA030 1920 RA002 2054 BA028 2178 BA021 2196 BA0272212 BA029 2267 BA023 2285 BA024 2340 BA022 2386 BA019 2421 BA015 2441BA020 2486 BA025 2827

TABLE 14 AUC values for anti-CD73 antibodies in FIGS. 5B-5P Antibody AUCRA002 61111 BA030 68786 BA026 72777 RA001 77155 BA028 90740 BA023 97165BA029 97805 BA024 99898 BA027 101503 BA019 102780 BA025 103903 BA021108065 BA015 112756 BA020 113013 BA022 123732

6.2.4 Inhibition of CD73 Enzymatic Activity by Anti-CD73 Antibodies

The ability of the humanized anti-CD73 antibodies to inhibit theenzymatic activity of CD73 was assessed using the same method asdescribed in Example 6.1.4. BA015, RA001, and RA002 were also examinedfor comparison.

As shown in FIGS. 6A-6P, each of the tested humanized antibodies,BA019-BA030, as well as chimeric antibody BA015 and the two referenceantibodies, but not the isotype control, inhibited the enzymaticactivity of CD73 expressed on Jurkat cells. The calculated AUC valuesfor anti-CD73 antibodies in FIGS. 6B-6P are presented in Table 15.

TABLE 15 AUC values for anti-CD73 antibodies in FIGS. 6B-6P Antibody AUCBA021 301.6 BA025 317.5 BA015 327.5 BA019 329.2 BA024 360.8 BA028 368.4BA020 377.8 RA002 384.4 BA022 385.8 BA026 386.6 BA023 386.7 BA030 396.9BA029 400.5 RA001 436.4 BA027 442.2

Additionally, the ability of the humanized antibodies to inhibit CD73enzymatic activity was assessed using a biochemical assay. Specifically,a recombinant human CD73 fused with a 6×His-tag (R&D Systems, 5795EN)was cross-linked to white nickel-coated 96-well plates overnight at 4°C. in an assay buffer (25 mM Tris, pH 7.5, 5 mM MgCl₂, and 0.005%Tween-20). The wells were gently washed twice, and an eight-point dosetitration of an anti-CD73 antibody or isotype control antibody from0.005 to 10 μg/ml was added at a final volume of 50 μl/well. The sampleswere incubated for 1 hour at 37° C. and 5% CO₂, and 50 μl of 200 μM AMPand 6 μM ATP was added to each well. Following a 20-minute incubation at37° C. and 5% CO₂, 100 μl of reconstituted CellTiter Glo® (Promega) wasadded to each well, and luminescence was recorded using Envisioninstrument (Perkin Elmer) after 5 minutes. BA015, RA001, and RA002 werealso examined for comparison.

Similar to the cell-based assay described above, in this biochemicalassay, the luminescence levels correlate with the CD73 activity in eachsample, because AMP inhibits the luciferase reaction, and hydrolysis ofAMP by CD73 relieves the inhibition. Thus, as shown in FIGS. 7A-7P, eachof the tested humanized antibodies, BA019-BA030, as well as chimericantibody BA015 and the two reference anti-CD73 antibodies, but not theisotype control, inhibited the enzymatic activity of recombinant CD73.The calculated AUC values for anti-CD73 antibodies in FIGS. 7B-7P arepresented in Table 16.

TABLE 16 AUC values for anti-CD73 antibodies in FIGS. 7B-7P Antibody AUCBA025 66.11 BA021 71.93 BA022 82.64 RA001 85.1 BA027 85.87 BA029 88.63BA015 91.2 BA026 95.04 BA020 95.3 BA019 98.11 BA024 110.8 BA023 116BA030 116.1 BA028 124.5 RA002 171.3

6.2.5 Anti-CD73 Antibodies Mediating CD73 Internalization

Internalization of humanized anti-CD73 antibodies into cells wasanalyzed using the cell toxicity assay as described in Section 6.1.5.

As shown in FIGS. 8A-8M, incubation with each of BA019-BA027 and BA030,along with αHFc-NC-MMAE, caused cell death of CD73-expressing Jurkatcells in a dose-dependent manner. This result suggested dose-dependentinternalization of these anti-CD73 antibodies upon CD73 binding.

6.3 Example 3: Generation and Characterization of Anti-CD73-TGFβ TrapFusion Proteins

This example describes the generation and characterization of antibodiescomprising a CD73-binding portion and a TGFβ-binding portion (“TGFβtrap”). These antibodies are also referred to as anti-CD73-TGFβ trapfusion proteins in this Example and Example 4.

6.3.1 Generation of Anti-CD73-TGFβ Trap Fusion Proteins

Exemplary anti-CD73-TGFβ trap fusion proteins comprise a firstpolypeptide comprising, from the N-terminus to the C-terminus, a heavychain of an anti-CD73 antibody described herein, a peptide linker, andan extracellular domain of TGFβR2; and a second polypeptide comprising alight chain of the anti-CD73 antibody. The sequences of exemplarypeptide linkers, the “TGFβ trap” portion, and anti-CD73-TGFβ trap fusionproteins are disclosed in Table 6.

In particular, an anti-CD73-TGFβ trap fusion protein designated BA035comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:113 and a light chain comprising the amino acid sequence of SEQ ID NO:92. Another anti-CD73-TGFβ trap fusion protein designated BA036comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:114 and a light chain comprising the amino acid sequence of SEQ ID NO:97. A third exemplary anti-CD73-TGFβ trap fusion protein designatedBA037 comprises a heavy chain comprising the amino acid sequence of SEQID NO: 115 and a light chain comprising the amino acid sequence of SEQID NO: 97. BA035 and BA037 were examined in this Example and Example 4.In the following experiments, two additional antibodies were used forcomparison: (i) BA012, an anti-CD73 antibody having the same sequencesas the anti-CD73 portion of BA035, was used to represent the activity ofthe CD73-binding portion of BA035; and (ii) isotype-TGFβ trap, anisotype control antibody comprising the peptide linker and theextracellular domain of TGFβR2, was used to represent the activity ofthe TGFβ-binding portions of BA035 and BA037. An isotype controlantibody without the peptide linker or the extracellular domain ofTGFβR2 was used to represent the baseline. BA035 and correspondingisotype controls were in the mouse IgG2a format with a N297Asubstitution in the heavy chain constant region. BA037 and correspondingcontrols were humanized antibodies in a human IgG1 format with N297Amutation.

6.3.2 Binding of Anti-CD73-TGFβ Trap Fusions Protein to CD73

The binding affinity of BA037 for recombinant CD73 was determined bysurface plasmon resonance (SPR) using the BIAcore® T200 system (GEHealthcare). BA037 (human anti-CD73-TGFβ trap fusion protein), BA025 (ananti-CD73 antibody having the same sequences as the anti-CD73 portion ofBA037), or an isotype-TGFβ trap were captured on a CM5 sensor chip (GEHealthcare, Series Sensor Chip CM5, 29-1496-03) using chip-immobilizedmouse anti-human IgG (GE Healthcare, Human Antibody Capture Kit,BR100839). Specifically, antibodies diluted in a running buffer (10 mMHEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) were capturedusing a 30-sec injection at a flow rate of 10 μl/min to reach capturelevels optimal for kinetic analysis (about 55 RU for BA025 and about 60RU for BA037 and isotype-TGFβ trap). The concentration of antibody toreach the optimal capture level was determined separately for eachexperiment. Recombinant human, cynomolgus, or mouse CD73 protein (R&DSystems, 5795EN; Sino Biological, 90192-C08H; and Sino Biological,50231-M08H, respectively), diluted in the running buffer at theconcentration of about 0.098 to 100 nM, were applied to the chip surfaceat a flow rate of 100 μl/min with a 3-min association phase and a 20-mindissociation phase. The sensor chip was regenerated between cycles withtwo 30-sec injections of 3M MgCl₂. Kinetic parameters were determinedusing a Langmuir 1:1 binding model using BIAcore® T200 Evaluationsoftware v3.0 (GE Healthcare). The analysis assumed that each Fab arm ofthe antibody interacted with one dimeric CD73 molecule. Data quality wasverified by visually inspecting deviations and curve fitting, and byevaluating the parameters of U-value, Rmax, Chi2, and Tc. The bindingkinetics (K_(a), K_(d) and K_(D)) were determined from the sensorgramanalyses and are shown in Table 17. Values are the geometric mean ofthree experiments. No measurable binding was observed for mouse CD73 toeither BA037 or BA025. No measurable binding was observed for CD73 toisotype-TGFβ trap.

TABLE 17 SPR-based affinity measurements for BA025 and BA037 binding toCD73 Human CD73 Cynomolgus CD73 K_(a) K_(d) K_(D) K_(a) K_(d) K_(D)Clone (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) BA037 4.79E+06 3.31E−04 0.0691.97E+06 2.40E−04 0.122 BA025 3.87E+06 3.23E−04 0.083 1.64E+06 2.33E−040.143

6.3.3 Binding of Anti-CD73-TGFβ Trap Fusions Protein to TGFβ

The binding affinity of BA037 for recombinant TGFβ was determined bysurface plasmon resonance (SPR) using the BIAcore® T200 system (GEHealthcare). BA037, BA025, or an isotype-TGFβ trap were captured on aCM5 sensor chip (GE Healthcare, Series Sensor Chip CM5, 29-1496-03)using chip-immobilized mouse anti-human IgG (GE Healthcare, HumanAntibody Capture Kit, BR100839). Specifically, antibodies diluted in arunning buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05%surfactant P20) were captured using a 30-sec injection at a flow rate of10 μl/min to reach capture levels optimal for kinetic analysis (about110 RU for BA025 and about 120 RU for BA037 and isotype-TGFβ trap). Theconcentration of antibody to reach the optimal capture level wasdetermined separately for each experiment. Recombinant human TGFβ1,TGFβ2, or TGFβ3 protein (Peprotech, AF-100-21C; Peprotech, 100-35B;Peprotech, AF-100-36E, respectively) were used in this example. EachTGFβ was diluted in the running buffer at concentrations of 0.0312 to 32nM and were flowed over the chip surface at a flow rate of 100 μl/minwith a 3-min association phase and a 20-min dissociation phase. Thesensor chip was regenerated between cycles with two 30-sec injections of3M MgCl₂. Kinetic parameters were determined using a Langmuir 1:1binding model using BIAcore® T200 Evaluation software v3.0 (GEHealthcare). Data quality was verified by visually inspecting deviationsand curve fitting, and by evaluating the parameters of U-value, Rmax,Chi2, and Tc. The binding kinetics (K_(a), K_(d) and K_(D)) weredetermined from the sensorgram analyses and are shown in Table 18.Values are the geometric mean of three experiments.

TABLE 18 SPR-based affinity measurements for BA037 and BA025 binding toTGFβ Human TGFβ1 Human TGFβ2 Human TGFβ3 K_(a) K_(d) K_(a) K_(d) K_(a)K_(d) Clone (1/Ms) (1/s) K_(D) (1/Ms) (1/s) K_(D) (1/Ms) (1/s) K_(D)BA037 9.66E+07 2.05E−04 2.1 pM 5.54E+07 8.85E−02 1.6 nM 5.00E+072.09E−04 4.2 pM isotype- 7.01E+07 9.63E−05 1.4 pM 5.67E+07 1.78E−01 1.5nM 6.16E+07 1.40E−04 2.3 pM TGFβ trap

6.3.4 Binding of Anti-CD73-TGFβ Trap Fusion Proteins to CD73-ExpressingCells

The affinity of BA035 for membrane-associated CD73 was assessed.Specifically, CHO cells were transduced with lentiviral vectorscomprising a full-length mouse CD73 coding sequence under the control ofa CMV promoter, and stable CHO-muCD73 cells were generated by antibioticselection. Expression of mouse CD73 was verified by flow cytometry usingpositive control antibodies. The stable CHO-muCD73 cells were grown insuspension culture in RPMI media supplemented with 10% heat-inactivatedFBS at 37° C. and 5% CO₂. For binding analysis, the stable CHO-muCD73cells were incubated for 30 minutes at 4° C. with an eight-point dosetitration of BA035, BA012, isotype-TGFβ trap, orisotype control antibodyfrom 0.005 to 10 μg/ml diluted in FACS buffer (PBS, 2 mM EDTA, 0.5% BSA,pH 7.2). The cells were washed twice in FACS buffer and incubated withFITC-conjugated mouse anti-human kappa detection antibody (LifeTechnologies, HP6062, 1:100 dilution in FACS buffer) for 30 minutes at4° C. The cells were then washed twice and analyzed using theLSRFortessa™ flow cytometer (BD Biosciences). FACS data were analyzedusing FACS DIVA and WEHI Weasel software, and were plotted with GraphpadPrism software.

As shown in FIG. 9A, BA035 retained its ability to bind to CD73,suggesting that fusion with the TGFβ trap did not abrogate theCD73-binding ability of anti-CD73 antibodies.

The binding affinity of BA035 to CD73 was further assessed in thepresence of TGFβ. Specifically, a ten-point dose titration ofrecombinant human TGFβ1 (Peprotech) from 0.002 to 40 μg/ml were added tostable CHO-muCD73 cells in concert with 10 μg/ml of BA035, BA012, orisotype control antibody. After a 30-minute incubation at 37° C. and 5%CO₂, the cells were washed twice in FACS buffer and incubated withFITC-conjugated mouse anti-human kappa detection antibody (LifeTechnologies, HP6062, 1:100 dilution in FACS buffer) for 30 minutes at4° C. The cells were then washed twice and analyzed using theLSRFortessa™ flow cytometer (BD Biosciences). FACS data were analyzedusing FACS DIVA and WEHI Weasel software, and were plotted with GraphpadPrism software.

As shown in FIG. 9B, BA035 retained its ability to bind to CD73 in thepresence of TGFβ1.

In a separate experiment, the affinity of BA037 for membrane-associatedCD73 from primary human, cynomolgus monkey, or mouse CD8⁺ T cells wasanalyzed (for the mouse cells, expression of mouse CD73 was verified byflow cytometry using positive control antibodies). Specifically,cryopreserved PBMCs and splenocytes were thawed in RPMI-1640 media. Forbinding analysis, PBMCs or splenocytes were incubated for 30 minutes at4° C. with a twelve-point dose titration of BA037, BA025, isotype-TGFβtrap, or isotype control antibody, each conjugated to PE, from 0.004 to666 nM diluted in assay buffer (PBS, 0.5% BSA, 1:100 Trustain FcX™).Cells also were stained with Zombie NIR™, fluorescently labeledanti-CD3, and fluorescently labeled anti-CD8 to identify viable CD8⁺ Tcells. The cells were then washed twice and analyzed using theLSRFortessa™ flow cytometer (BD Biosciences). FACS data were analyzedusing WEASEL software, and were plotted with Graphpad Prism software.FIGS. 13A and 13B show the human and cynomolgus monkey T cell bindingdata obtained from this experiment.

As shown in FIGS. 13A and 13B, BA037 demonstrated dose-dependent bindingto human and cynomolgus monkey CD8⁺ T cells, which was broadly similarto that observed for BA025 (the anti-CD73 component of BA037). BA037binding curves to human and cynomolgus monkey CD8⁺ T cells werecomparable to each other with average cellular EC₅₀ values of 2.57 and9.67 nM respectively. In contrast, no binding was observed with isotypecontrol antibodies (with or without a TGFβRII trap fusion). No bindingof BA037 to mouse CD8⁺ T cells was observed (data not shown).

6.3.5 Inhibition of CD73 Enzymatic Activity by Anti-CD73-TGFβ TrapFusion Proteins

The ability of BA035 to inhibit CD73 enzymatic activity was assessedusing a biochemical assay. Specifically, 0.3 μg/ml of recombinant human,cynomolgus, or mouse CD73 protein fused with a 6×His-tag (R&D Systems,5795EN; Sino Biological, 90192-C08H; or R&D Systems, 4488EN,respectively) was mixed with an eight-point dose titration of BA035,BA012, isotype-TGFβ trap, or isotype control antibody from 0.005 to 10μg/ml in an assay buffer (25 mM Tris pH 7.5, 5 mM MgCl₂, and 0.005%Tween-20) to reach a final volume of 50 μl/well in a whitetissue-culture treated plate. The samples were incubated for 30 minutesat 37° C. and 5% CO₂, and 50 μl of 200 μM AMP and 6 μM ATP was added toeach well. Following a 20-minute incubation at 37° C. and 5% CO₂, 100 μlof reconstituted CellTiter Glo® (Promega) was added to each well, andluminescence was recorded using Envision instrument (Perkin Elmer) after5 minutes. The data were plotted with Graphpad Prism software.

As shown in FIGS. 9C and 9D, BA035 retained its ability to inhibit CD73activity, suggesting that fusion with the TGFβ trap did not abrogate theCD73-inhibiting function of anti-CD73 antibodies.

The ability of BA035 to inhibit CD73 activity was further assessed inthe presence of TGFβ. Specifically, 40 μg/ml of recombinant human TGFβ1(Peprotech) was mixed with 0.3 μg/ml of recombinant mouse CD73 proteinand an eight-point dose titration of BA035, BA012, isotype-TGFβ trap, orisotype control antibody from 0.009 to 6.6 μg/ml to reach a final volumeof 50 μl (final TGFβ1 concentration to be 20 μg/ml). The samples wereincubated for 30 minutes at 37° C. and 5% CO2, and 50 μl of 200 μM AMPand 6 μM ATP was added to each well. Following a 20-minute incubation at37° C. and 5% CO2, 100 μl of reconstituted CellTiter Glo® (Promega) wasadded to each well, and luminescence was recorded using Envisioninstrument (Perkin Elmer) after 5 minutes. The data were plotted withGraphpad Prism software.

As shown in FIG. 9E, BA035 retained its ability to inhibit CD73 activityin the presence of TGFβ1.

In a separate experiment, the ability of BA037 to inhibit plate-boundCD73 enzymatic activity was also assessed. Specifically, 50 μL of 300ng/ml recombinant human, cynomolgus, or mouse CD73 protein fused with a6×His-tag (R&D Systems, 5795EN; Sino Biological, 90192-C08H; or, SinoBiological, 50231-M08H, respectively) was incubated in each well of aNi-coated white flat-bottom 96-well plate for 2 hours at 37° C. Afterwashing twice, 50 μL/well of BA037, BA025, isotype-TGFβ trap, or isotypecontrol antibody were added to each well. For human and cynomolgusmonkey CD73, a ten-point concentration curve from 0.001 to 20 nM ofantibody in an assay buffer was used. For mouse CD73, an eight-pointconcentration curve from 0.01 to 20 nM of antibody in an assay bufferwas used. The samples were incubated for 60 minutes at 37° C. and 5%CO₂, and 50 μL of 200 μM AMP and 6 μM ATP was added to each well.Following a 20-minute incubation at 37° C. and 5% CO₂, 100 μl ofreconstituted CellTiter Glo® (Promega) was added to each well, andluminescence was recorded using EnVision instrument (Perkin Elmer) after5 minutes. The data were plotted with Graphpad Prism software.

The ability of BA037 to inhibit soluble CD73 enzymatic activity wasassessed using a biochemical assay. Specifically, 0.3 μg/ml ofrecombinant human, cynomolgus, or mouse CD73 protein fused with a6×His-tag (R&D Systems, 5795EN; Sino Biological, 90192-C08H; or, SinoBiological, 50231-M08H, respectively) was mixed with an eleven-pointdose titration of BA037, BA025, isotype-TGFβ trap, or isotype controlantibody from 0.005 to 100 nM in an assay buffer (25 mM Tris pH 7.5, 5mM MgCl₂) to reach a final volume of 50 μl/well in a whitetissue-culture treated plate. The samples were incubated for 60 minutesat 37° C. and 5% CO₂, and 50 μl of 200 μM AMP and 6 μM ATP was added toeach well. Following a 20-minute incubation at 37° C. and 5% CO₂, 100 μlof reconstituted CellTiter Glo® (Promega) was added to each well, andluminescence was recorded using EnVision instrument (Perkin Elmer) after5 minutes. The data were plotted with Graphpad Prism software.

The ability of BA037 to inhibit cell-expressed CD73 enzymatic activitywas also assessed. Jurkat cells expressing human CD73 (or control cellsnot expressing CD73) were used in this experiment. Cells at aconcentration of 1×10⁶ cells/mL were plated in a flat bottom clear96-well cell culture plate in a volume of 50 μL/well. 50 μL/well ofBA037, BA025, isotype-TGFβ trap, or isotype control antibody were thenadded to each well in a ten-point concentration curve from 0.001 to 20nM of antibody in an assay buffer. The samples were incubated for 60minutes at 37° C. and 5% CO₂, and 50 μL of 200 μM AMP and 6 μM ATP wasadded to each well. Following a 20-minute incubation at 37° C. and 5%CO₂, 100 μl of reconstituted CellTiter Glo® (Promega) was added to eachwell, and luminescence was recorded using EnVision instrument (PerkinElmer) after 5 minutes. The data were plotted with Graphpad Prismsoftware.

As shown in FIGS. 14A, 14B, 15A and 15B, BA037 demonstrateddose-dependent inhibition of the enzymatic activity of both human andcynomolgus monkey recombinant CD73 in plate-bound format with averageIC₅₀ values of 0.518 nM and 0.707 nM, respectively. BA025 similarlyinhibited CD73 enzymatic activity across species, whereas the isotypecontrol antibody and the isotype-TGFβ trap fusion protein did not,thereby confirming that the inhibition effected by BA037 was entirelydue to the anti-CD73 portion of the molecule. As expected, BA037 did notblock the enzymatic activity of mouse recombinant CD73 (data not shown),because of its low affinity for mouse CD73. In contrast, an anti-mouseCD73 antibody (Clone TY/11.8) inhibited recombinant mouse CD73 activityin a dose-dependent manner (data not shown).

As shown in FIGS. 14C, 14D, 15C and 15D, BA037 demonstrateddose-dependent inhibition of the enzymatic activity of both human andcynomolgus monkey recombinant CD73 in soluble form, with average IC₅₀values of 0.99 nM and 0.623 nM, respectively.

As shown in FIGS. 16A and 16B, BA037 also inhibited the enzymaticactivity of human CD73 produced by Jurkat cells engineered tooverexpress human CD73, with an average IC₅₀ value of 0.235 nM.

6.3.6 Neutralization of TGFβ Signaling and Mesenchymal AntigenExpression by Anti-CD73-TGFβ Trap Fusion Proteins

The ability of BA035 to impair TGFβ-induced signaling was evaluatedusing an ectopically expressed reporter system and an endogenousreporter system.

The ectopically expressed reporter was a vector comprising a luciferasecoding sequence under control of a SMAD binding element (SBE) in acommercial HEK293 cell line (BPS Biosciences). Briefly, HEK293 cellswere transfected with the vector, thereby generating HEK293-SMADreporter cells. The HEK293-SMAD reporter cells were plated at thedensity of 3.5×10⁴ cells/well and cultured overnight in 100 μl of growthmedium (MEM medium supplemented with 10% FBS) in white flat-bottomtissue culture plate at 37° C. and 5% CO₂. The growth media was replacedwith assay media (MEM medium supplemented with 0.5% FBS), and the cellswere cultured for 4.5 hours at 37° C. and 5% CO₂. An eight-point dosetitration of BA035, BA012, isotype-TGFβ trap, or isotype controlantibody from 0.009 to 20 μg/ml was added to the cells in concert with20 ng/ml of recombinant human TGFβ (Peprotech), and the cells wereincubated overnight at 37° C. and 5% CO₂. To detect TGFβ-inducedluciferase activity, 100 μl of reconstituted Bio-Glo™ (Promega) wasadded to each well, and luminescence was recorded using an Envisioninstrument (Perkin Elmer) after 5 minutes.

As shown in FIG. 10A, both BA035 and isotype-TGFβ trap, but not BA012,showed a dose-dependent inhibition of the TGFβ-induced luciferaseexpression in the HEK293-SMAD reporter cells.

In a separate experiment, an eleven-point dose titration of BA037,BA025, isotype-TGFβ trap, or isotype control antibody from 0.0003 to 20nM was conducted separately for TGFβ1, TGFβ2, and TGFβ3 isoforms.Otherwise the same protocol was used as above. As shown in FIGS.17A-17C, both BA037 and isotype-TGFβ trap, but not BA025, showed adose-dependent inhibition of the TGFβ-induced luciferase expression forthe β1 and β3 isoforms, but not the β2 isoform, in the HEK293-SMADreporter cells.

To determine the effect of these molecules on epithelial-to-mesenchymaltransition (EMT) in a tumor cell line, a red fluorescent protein (RFP)coding sequence was inserted at the 3′ end of the endogenous openreading frame of the vimentin gene in the genome of lung carcinoma A549cells (ATCC® CCL-185EMT™). This A549-Vimentin-RFP cell reporter systemallowed detection of TGFβ-induced expression of vimentin, a marker ofEMT, by measuring fluorescence from RFP fused to the C-terminus of theVimentin protein. Briefly, A549-Vimentin-RFP cells were grown insuspension culture in F-12K media supplemented with 10% heat-inactivatedFBS at 37° C. and 5% CO₂, and were incubated with 10 μg/ml BA035, BA012,isotype-TGFβ trap, or isotype control antibody in the presence orabsence of 2.5 ng/ml TGFβ1 for 48 hours. The cells were washed twice inFACS buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) and analyzed using theLSRFortessa™ flow cytometer (BD Biosciences). FACS data were analyzedusing FACS DIVA and WEHI Weasel software and were plotted with GraphpadPrism software.

As shown in FIG. 10B, BA035 and isotype-TGFβ trap, but not BA012 orisotype control, inhibited TGFβ1-induced upregulation of vimentinexpression levels.

In a separate set of experiments, an eleven-point dose titration ofBA037, BA025, isotype-TGFβ trap, or isotype control antibody from 0.0028to 165 nM was conducted separately for TGFβ1, TGFβ2, and TGFβ3 isoforms.A549-Vimentin-RFP cells were grown and prepared as described above. TGFβwas preincubated with antibodies for 20 minutes at room temperature(FIG. 18A-18F) or 30 minutes at 37° C. (FIG. 18G-18I). The antibody andTGFβ were then added to cells and incubated for 24 hours at 37° C. Cellswere washed with PBS and analyzed by flow cytometry using the BDFACSCanto II™ flow cytometer. FACS data were analyzed using FACS DIVAsoftware and were plotted with Graphpad Prism software. As shown inFIGS. 18A-18I, BA037 and isotype-TGFβ trap, but not BA025 or isotypecontrol, inhibited TGFβ1- and TGFβ3-induced upregulation of vimentinexpression levels.

6.3.7 Internalization of Anti-CD73-TGFβ Trap Fusion Proteins Upon CD73Binding

Internalization of the anti-CD73-TGFβ trap, BA037, or control antibodiesinto cells expressing CD73 was analyzed using a cell toxicity assay. Thecell toxicity assay employed an antibody-drug conjugate αHFc-NC-DM1(anti-human IgG Fe antibody conjugated to maytansinoid DM1 (DM1), with anon-cleavable linker, Moradec LLC), which could transport the cytotoxicpayload DM1 into the cytoplasm upon internalization. Briefly, a Jurkatcell line ectopically expressing human CD73, as described in Section6.1.4, was plated in white flat-bottom tissue culture plates at adensity of 1×10⁴ per well. A seven-point dose titration of an anti-CD73antibody or isotype control antibody from 0.03 to 20 nM, along withαHFc-NC-DM1 at the same concentration as the antibody, was added to thecells to reach a final volume of 100 μl/well. Following a 72-hourincubation at 37° C. and 5% CO₂, 100 μl of reconstituted CellTiter Glo®(Promega) was added to each well. After 5 minutes, luminescence,representing cell viability, was recorded using Envision instrument(Perkin Elmer).

As shown in FIGS. 19A and 19B, incubation with BA025 or BA037, alongwith αHFc-NC-DM1, caused cell death of CD73-expressing Jurkat cells in adose-dependent manner. As shown in FIGS. 19C and 19D, controlexperiments without αHFc-NC-DM1 did not result in cell death. Thisresult showed that the presence of a TGFβ cytokine trap moiety in BA037did not affect its CD73-mediated internalization.

6.3.8 Anti-CD73-TGFβ Trap Fusion Proteins do not Activate FcγR

Activation of the high-affinity allotypes FcγRIIA (H131) or FcγRIIIA(V158) by anti-CD73-TGFβ trap fusion protein (BA037) or controlantibodies was measured, in the presence of cells expressing CD73, usingJurkat reporter cell lines engineered to express either FcγRIIA (H131)or FcγRIIIA (V158) together with a downstream luciferase 2 (luc2)reporter gene driven by an NFAT promoter. Briefly, 25 μL of CHO cellsexpressing CD73 (CHO-CD73, at 2.4×10⁶ cells/mL) were dispensed into thewells of a 96-well, flat-bottom, white assay plate, resulting in 6.0×10⁴cells per well. A ten-point dose titration (from 0.0005 to 10 nM) ofBA037, BA025, an Fc-competent anti-CD73, isotype-TGFβ trap or isotypecontrol antibody was added to the cells. Next, 25 μL of either theJurkat FcγRIIA (H131) or Jurkat FcγRIIIA (V158) reporter cell suspension(at 6×10⁶ cells/mL, resulting in 1.5×10⁵ cells/well) was added to thewells. Following a 20-hour incubation at 37° C. and 5% CO₂, 75 μL ofreconstituted CellTiter Glo® (Promega) was added to each well, andluminescence, representing cell viability, was recorded using Envisioninstrument (Perkin Elmer) after 5 minutes.

In these experiments, BA037 and BA025 showed no appreciable activationof high affinity FcγRIIA (H131) or FcγRIIIA (V158) signaling upon targetengagement (FIGS. 20A and 20B, dark circles and squares). By contrast,an Fc-competent anti-CD73 antibody (human IgG1λ) induced robust reportergene activation (FIGS. 20A and 20B, dark diamonds). These findings areconsistent with BA037 lacking associated Fc-mediated effector cellactivity.

6.4 Example 4: In Vivo Activities of Anti-CD73 Antibodies

This example describes the anti-tumor activities of anti-CD73antibodies, including anti-CD73-TGFβ trap fusion proteins, in variousmodels of tumor growth and metastasis.

6.4.1 Anti-CD73 Antibodies Impairs Tumor Growth and Metastasis

The anti-tumor function of anti-CD73 antibodies was tested in animalmodels of colon cancer and tumor metastasis.

In the colon cancer model, 6-8 week old female Balb/c mice were injectedwith 5×10⁴ CT26 tumor cells subcutaneously on day 0, and treatedintraperitoneally with 200 μg of BA012, an antibody comprising thevariable domain of RA001 and a murine IgG2a constant domain comprisingan N297A mutation (designated RA001-IgG2a-N207A), or an isotype controlantibody, on days 5, 9, and 14. Tumor growth was monitored bi-weeklyusing a digital caliper. Tumor volume was calculated using the formulaof volume=height×width×0.52.

As shown in FIG. 11A, BA012 inhibited tumor growth more potently thanthe chimeric variant of RA001.

In the tumor metastasis model, 6-8 week old female Balb/c mice wereinjected intravenously on day 0 with 2×10⁴ EMT6 breast carcinoma cells,which expressed high levels of endogenous CD73. 200 μg of BA012 orisotype control antibody were administered intraperitoneally on the sameday (day 0) and on day 3. The mice were euthanized on day 14, andmetastatic nodules in the lungs were evaluated by washing the tissues inPBS, staining for contrast using 4% India ink, and fixing at roomtemperature using Fekete's solution (aqueous solution of ethanol,formaldehyde, and acetic acid). White metastatic nodules were countedmanually and plotted with Graphpad Prism software.

As shown in FIG. 11B, BA012 inhibited invasion of the EMT6 tumor cellsinto the lungs relative to the isotype control, suggesting that theanti-CD73 antibody inhibited tumor metastasis.

In a parallel experiment, 6-8 week old female Balb/c mice were injectedintravenously on day 0 with 2×10⁴ EMT6 breast carcinoma cells. 400 μg ofBA012, 400 μg isotype control antibody or 492 μg (equimolar to BA012 andisotype control) of BA035 were administered intraperitoneally on thesame day (day 0) and on day 3. The mice were euthanized on day 14, andmetastatic nodules in the lungs were evaluated by washing the tissues inPBS, staining for contrast using 4% India ink, and fixing at roomtemperature using Fekete's solution (aqueous solution of ethanol,formaldehyde, and acetic acid). White metastatic nodules were countedmanually and plotted with Graphpad Prism software.

As shown in FIG. 21 , BA035 inhibited invasion of the EMT6 tumor cellsinto the lungs relative to BA012 and the isotype control, demonstratingthat the anti-CD73-TGFβ trap fusion protein is capable of inhibitingtumor metastasis.

6.4.2 Anti-CD73-TGFβ Trap Fusion Proteins Show Synergy Between CD73Antagonism and TGFβ Neutralization in Tumor Inhibition

The functions of anti-CD73-TGFβ trap fusion proteins were tested in twoanimal models of tumor growth.

In the first model, 6-8 week old female C57BL/6 mice were injected with2×10⁵ HPV16-E6/7⁺c-Ha-ras⁺ TC1 mouse lung cancer cells subcutaneously onday 0. The tumor-bearing mice were randomized on day 5, and were treatedintraperitoneally with 200 μg of BA012 or isotype control antibody, or246 μg of BA035 or isotype-TGFβ trap on days 5, 8, and 11. Tumor growthwas monitored bi-weekly using a digital caliper. Tumor volume wascalculated using the formula of volume=height×width×width×0.52, and thedata were plotted with Graphpad Prism software.

As shown in FIG. 12A, BA035, but not BA012 or isotype-TGFβ trap alone,inhibited tumor growth.

In a second model, of LLC Lewis lung cancer, wildtype female C57BL/6mice were implanted with 0.5×10⁶ LLC tumor cells (ATCC no. CRL-1642).Dosing started when tumors reached 75-100 mm³, which occurred on day 4.Mice were administered 200 μg isotype control antibody, 246 μg of BA035,200 μg of BA012, or 246 μg of isotype-TGFβ trap on days 4, 7, and 11.Tumor growth was monitored bi-weekly using a digital caliper. Tumorvolume was calculated using the formula ofvolume=height×width×width×0.52, and the data were plotted with GraphpadPrism software.

As shown in FIG. 22 , BA035 was more effective than BA012 orisotype-TGFβ trap fusion protein alone at inhibiting tumor growth.

In a third model, a Detroit562 mouse xenograft model that is known to besensitive to TGFβ inhibition was employed (see Koopman et al., CancerRes. 68, 561-569 (2008)). Specifically, Detroit562 tumor cells wereadministered subcutaneously to 6-8 week old female nude (Foxn1^(nu))mice (1×10⁶ cell/mouse). Tumor-bearing mice (n=8-10 mice/group) weretreated intraperitoneally (i.p.) with 246 μg of BA035, 200 μg of BA012,246 μg of isotype-TGFβ trap, a combination of 200 μg of BA012 and 246 μgof isotype-TGFβ trap, or 200 μg of isotype control on days 8, 11, and 15post-tumor implantation. Tumor growth was monitored bi-weekly using adigital caliper. Tumor volume was calculated using the formulavolume=height×width×width×0.52, and the data were plotted with GraphpadPrism software.

As shown in FIG. 23 , all treatments comprising a TGFβ trap moiety wereeffective in this model.

In a fourth model, 6-8 week old female Balb/c mice were injected with4×10⁵ EMT6 tumor cell subcutaneously on day 0. The tumor-bearing micewere randomized and treated intraperitoneally with 246 μg of BA035, 200μg of BA012, 246 μg of isotype-TGFβ trap, a combination of 200 μg ofBA012 and 246 μg of isotype-TGFβ trap, or 200 μg of isotype controlantibody on days 5, 8, and 11. Tumor growth was monitored bi-weeklyusing a digital caliper. Tumor volume was calculated using the formulaof volume=height×width×width×0.52, and the data were plotted withGraphpad Prism software.

As shown in FIG. 12B, each of the antibody treatments, except for theisotype control antibody, inhibited tumor growth. BA035 showed strongeranti-tumor activity compared to the combination of BA012 andisotype-TGFβ trap, indicating synergy between CD73 antagonism and TGFβneutralization when the two activities are provided in a single complex.

6.4.3 Anti-CD73-TGFβ Trap Fusion Proteins Effectively Combine with OtherCancer Treatments

The ability of anti-CD73-TGFβ trap fusion proteins to enhance theefficacy of other cancer treatment was tested in two animal models oftumor growth.

Using the EMT6 model described above, groups of mice were treated withBA012, isotype control, BA035, isotype-TGFβ trap, or BA012 plusisotype-TGFβ trap. Antibodies were dosed on days 7, 10, 14 and 17. Micewere then treated with either doxorubicin or an anti-PD-1 antibody. Acontrol group did not receive combination treatment. Doxorubicin wasadministered intravenously on days 7 and 14 at 400 μg per injection.Anti-PD-1 antibody (clone RMP1-14, BioXcell) was administered at 200 μgper injection at the same time points as the other antibodies.

As shown in FIGS. 24A-24D, all treatments demonstrated some effect atinhibiting tumor growth relative to isotype control. Notably, thecombination of BA035 with an anti-PD-1 antibody was more effective atinhibiting tumor growth than the combination of anti-PD-1 antibody witheither BA012 or an isotype-TGFβ trap, as shown in FIG. 24B. Similarly,as shown in FIGS. 24C and 24D, the combination of BA035 with doxorubicinwas more effective at inhibiting tumor growth than the combination ofdoxorubicin with either BA012 or an isotype-TGFβ trap.

In a colon cancer model, 6-8 week old female Balb/cJ mice were injectedwith 5×10⁴ CT26 tumor cells subcutaneously on day 0. Once tumors reachedan average size of 75-100 mm³, mice were treated intraperitoneally with200 μg BA012, 200 μg of an isotype control antibody, 246 μg of anisotype-TGFβ trap, 246 μg BA035, or 200 μg BA012 plus 200 μg of anisotype-TGFβ trap, on days 13, 17, and 20 after injection of the CT26cells. Mice were also injected with 800 μg of etoposideintraperitoneally on days 12 and 19. Tumor growth was monitored twiceweekly using a digital caliper. Tumor volume was calculated using theformula of volume=height×width×width×0.52. Mice were euthanized oncetumors reached 1000 mm³.

As shown in FIG. 25 , the combination of BA035 and etoposide was moreeffective at inhibiting tumor growth than etoposide alone, a combinationof etoposide and BA012, or a combination of etoposide and either of theisotype controls. The fusion protein BA035 was also more effective incombination with etoposide than was the combination of BA012 and anisotype-TGFβ trap in combination with etoposide.

6.5 Example 5: Epitope Mapping

This example describes the portions of CD73 bound by BA037.

6.5.1 Epitope Mapping by HDX

The interaction of human CD73 with the Fab fragment of BA037 (BA037-Fab)was studied by HDX mass spectrometry. These data were used to identifyepitope regions bound by BA037-Fab on human CD73. The interaction ofCD73 with BA037-Fab were evaluated using the methods below.

CD73 Interaction with Anti-Human CD73 Fab20 μL recombinant human CD73 (8 μg; R&D Systems, Cat. No. 5795-EN) or 20μL recombinant human CD73 and BA037-Fab mixture (8 μg:24 μg) wasincubated with 110 μL deuterium oxide labeling buffer (50 mM sodiumphosphate, 100 mM sodium chloride at pD 7.4) for 0 sec, 10 sec, 60 sec,600 sec, or 3600 sec at 20° C. Hydrogen/deuterium exchange was quenchedby adding 130 μL of 4 M guanidine hydrochloride, 0.85 M TCEP buffer(final pH 2.5) and incubating the mixture for 3 min at 20° C.Subsequently, the quenched samples were subjected to on columnpepsin/protease XIII digestion and LC-MS analysis, as described below.The mass spectra were recorded in MS only mode.

Pepsin/Protease XLI Digestion and LC-MS

Each CD73 or CD73:BA037-Fab mixture was subjected to on-columnpepsin/protease XIII digestion using an in-house packed pepsin/proteaseXIII (w/w, 1:1) column, and the resultant peptides were analyzed usingan UPLC-MS system comprised of a Waters Acquity UPLC coupled to a QExactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo). Thepeptides were separated on a 50 mm×1 mm C8 column with a 16.5 mingradient from 2-30% Solvent B (0.2% formic acid in acentonitrile) inSolvent A (0.2% formic acid in water). Peptide identification wasperformed by searching MS/MS data against the human CD73 sequence withMascot. The mass tolerance for the precursor and product ions was 7 ppmand 0.02 Da, respectively.

HDX Data Analysis

Raw MS data was processed using HDX WorkBench software for the analysisof H/D exchange MS data. The deuterium levels were calculated using theaverage mass difference between the deuterated peptide and its nativeform (to). For the calculation of deuterium incorporation, the massspectra for a given peptide were combined across the extracted ionchromatogram peak and the weighted average m/z was calculated. The massincrease from the mass of the native peptide (0 minute) to the weightedaveraged mass corresponds to the level of deuterium incorporation.

Epitope Binding of Anti-Human CD73 Fab

Residues contributing to the epitope were identified by protection fromdeuterium exchange when BA037-Fab was bound to CD73. All the residues inthis paragraph are numbered according to SEQ ID NO: 99. One regionconsisting of residues 144-170 (IKAKGPLASQISGLYLPYKVLPVGDEV, SEQ ID NO:90), experienced strong deuterium protection when human CD73 was boundto BA037-Fab. Thus, this region corresponds to an epitope or portionthereof of BA037 on CD73. Another region consisting of residues 195-213(DEITALQPEVDKLKTLNVN, SEQ ID NO: 91) also experienced strong deuteriumprotection when human CD73 was bound to BA037-Fab. Thus, this regionalso corresponds to an epitope or portion thereof of BA037 on CD73. Bothregions (residues 144-170 and 195-213 of SEQ ID NO: 129) may also becollectively considered an epitope or portion thereof of BA037 on CD73.

6.5.2 Epitope Mapping by Mutation of CD73

The binding of BA037 and RA001 to human and mouse CD73 were furtherstudied using a series of CD73 mutants, as described in Table 19 andTable 20. CD73 residues were selected for mutagenesis based ondifferences between human and mouse CD73 sequence or based onconsideration of the human CD73 crystal structure (PDB reference No.4H2F).

The affinity of BA037 and the reference anti-CD73 antibody, RA001, torecombinant CD73 proteins was determined by SPR using the BIAcore® T200system (GE Healthcare). BA037, RA001, or an isotype control-TGFβ trapfusion protein were captured on a CM5 sensor chip (GE Healthcare, SeriesSensor Chip CM5, 29-1496-03) using chip-immobilized mouse anti-human IgG(GE Healthcare, Human Antibody Capture Kit, BR100839). Specifically,antibodies diluted in a running buffer (10 mM HEPES, 150 mM NaCl, and0.05% surfactant P20) were captured using a flow rate of 10 μl/min toreach capture levels optimal for kinetic analysis. The concentration ofantibody to reach the optimal capture level was fixed to 1.5 μg/ml.Recombinant CD73 used in this example was produced in house. Binding tohuman wild-type CD73 was similar for the internal CD73 and a commercialrecombinant CD73 (Sino Biological Cat. No. 10904-H08H) (data not shown).CD73 was serially diluted and each mutant was flowed over the chipsurface at a flow rate of 50 μl/min with a 2-min association phase and a10-min dissociation phase. The sensor chip was regenerated betweencycles with one 90-sec injection of 3M MgCl₂. Kinetic parameters weredetermined using a Langmuir 1:1 binding model using BIAcore®T200Evaluation software v3.0 (GE Healthcare).

The effect of CD73 mutations on maximal binding of BA037 and RA001 isshown in Table 19. Maximal binding was measured at 60 nM. Y161A showed apartial loss of BA037 binding, as did a quadruple human-to-mousemutation (Y158F, Y161S, P165S, D168G). When the same four residues weremutated to alanine (Y158A, Y161A, P165A, D168A), additional BA037binding was lost. In contrast, RA001 bound at similar levels to all CD73mutants tested.

Consistent with prior observations, no significant binding was observedto wild-type mouse CD73 for BA037. However, mutation of residues 163,167, and 170 in mouse CD73 to the corresponding human residues (i.e.,the mouse-to-human mutations S163Y, S167P, G170D) resulted inrestoration of BA037 binding (as shown in Table 19) or partialrestoration of binding affinity (as shown in Table 20). Furthermore,mutation of residues 109, 111, 154, 160, 163, 167, 170, 200, 298, and299 in mouse CD73 to the corresponding human residues (i.e., themouse-to-human mutations I109A, G111R, H154S, F160Y, S163Y, S167P,G170D, S200T, D298E, K299R) resulted in restoration of both binding (asshown in Table 19) and binding affinity (as shown in Table 20). RA001bound to mouse CD73 at similar levels to human CD73.

TABLE 19 Effect of CD73 mutations on maximal BA037 and RA001 binding SEQID CD73 Variant NO: BA037 RA001 Human WT 99 + + Y158A 59 + + Y161A 60+/− + T198A 61 + + K274A 62 + + S269A 132 + + Y158A, Y161A, P165A, D168A133 − + Y158F, Y161S, P165S, D168G 134 +/− + (human to mouse) S152H,Y158F, Y161S 135 + + (human to mouse) P165S, D168G 136 + + (human tomouse) Mouse WT 102 − + I109A, G111R, H154S, F160Y, 137 + + S163Y,S167P, G170D, S200T, D298E, K299R (mouse to human) S163Y, S167P, G170D138 + + (mouse to human) +: at least 70% relative to maximal binding tohuman wild-type CD73 +/−: less than 70% and at least 20% relative tomaximal binding to human wild-type CD73 −: less than 20% relative tomaximal binding to human wild-type CD73

The effect of the foregoing CD73 mutations on the K_(A) (bindingaffinity) of BA037 and RA001 binding is shown in Table 20. In thisexperiment, the K_(A) values for BA037 and RA001 binding to wild-typehuman CD73 were determined to be 7.35×10⁹ M⁻¹ and 3.21×10⁹ M⁻¹,respectively.

TABLE 20 Effect of CD73 mutations on K_(A) of BA037 and RA001 bindingSEQ ID CD73 Variant NO: BA037 RA001 Human WT 99 + + Y158A 59 +/− + Y161A60 − + T198A 61 + + K274A 62 + + S269A 132 + + Y158A, Y161A, P165A,D168A 133 − + (human to mouse) Y158F, Y161S, P165S, D168G 134 − + (humanto mouse) S152H, Y158F, Y161S 135 − + (human to mouse) P165S, D168G 136− + (human to mouse) Mouse WT 102 − + I109A, G111R, H154S, F160Y,137 + + S163Y, S167P, G170D, S200T, D298E, K299R (mouse to human) S163Y,S167P, G170D 138 +/− + (mouse to human) +: no more than 4 times loweraffinity relative to affinity to human wild-type CD73 +/−: between 4 and10 times lower affinity relative to affinity to human wild-type CD73 −:at least 10 times lower affinity relative to affinity to human wild-typeCD73

As shown in Tables 19 and 20, mutation of one or more of residues Y158,Y161, P165, and/or D168 reduced maximal binding and affinity of BA037 tohuman CD73. BA037 was not sensitive to single mutation of residues T198,S269 or K274. Binding of RA001 was not affected by any mutation tested.These results indicate that BA037 binds to human CD73 via an epitope (orepitopes) comprising residues Y158, Y161, P165, and/or D168. Theseresults also indicate that the epitope of BA037 is different from theepitope of the reference antibody RA001.

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entiretiesand for all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

Other embodiments are within the following claims.

What is claimed:
 1. An isolated polynucleotide encoding a heavy chainvariable region or a light chain variable region of an antibody, whereinthe heavy chain variable region comprises complementarity determiningregions (CDRs) CDRH1, CDRH2, and CDRH3, and the light chain variableregion comprises complementarity determining regions CDRL1, CDRL2, andCDRL3, wherein: (a) CDRH1 comprises the amino acid sequence ofX₁X₂WX₃X₄(SEQ ID NO: 13), wherein X₁ is S or N; X₂ is S or Y; X₃ is I orM; and X₄ is N or H; (b) CDRH2 comprises the amino acid sequence ofX₁IYPRX₂X₃DTNYX₄X₅KFKX₆ (SEQ ID NO: 14), wherein X₁ is R or T; X₂ is N,A, or S; X₃ is G or S; X₄ is N, A, or S; X₅ is G or Q; and X₆ is D or G;(c) CDRH3 comprises the amino acid sequence of LLDYSMDY (SEQ ID NO: 7);(d) CDRL1 comprises the amino acid sequence of RASQDISX₁X₂LN (SEQ ID NO:16), wherein X₁ is N or I; and X₂ is Y or S; (e) CDRL2 comprises theamino acid sequence of YTSRLHS (SEQ ID NO: 10); and (f) CDRL3 comprisesthe amino acid sequence of QQGNTLPXT (SEQ ID NO: 17), wherein: X is L orW.
 2. The isolated polynucleotide of claim 1, wherein CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3 comprise the CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, and CDRL3 amino acid sequences, respectively, set forth in SEQ IDNOs: 2, 4, 7, 8, 10, and 12; 2, 5, 7, 8, 10, and 12; 1, 3, 7, 8, 10, and11; 2, 4, 7, 8, 10, and 11; 2, 4, 7, 9, 10, and 12; 2, 6, 7, 8, 10, and12; 2, 5, 7, 9, 10, and 12; or 2, 6, 7, 9, 10, and
 12. 3. The isolatedpolynucleotide of claim 2, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and CDRL3 comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3amino acid sequences, respectively, set forth in SEQ ID NOs: 2, 5, 7, 8,10, and
 12. 4. The isolated polynucleotide of claim 1, wherein: (a) theheavy chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 19-21 and 23-30; (b) the lightchain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 33-35 and 37-40; and/or (c) the heavychain variable region and the light chain variable region, respectively,comprise the amino acid sequences set forth in SEQ ID NOs: 24 and 37; 27and 37; 19 and 33; 20 and 34; 21 and 35; 23 and 37; 23 and 38; 24 and38; 25 and 37; 26 and 37; 28 and 37; 25 and 38; 26 and 38; 27 and 38; 28and 38; 29 and 37; 30 and 37; 23 and 39; or 23 and
 40. 5. The isolatedpolynucleotide of claim 4, wherein the heavy chain variable region andthe light chain variable region, respectively, comprise the amino acidsequences set forth in SEQ ID NOs: 27 and
 37. 6. The isolatedpolynucleotide of claim 1, wherein the antibody comprises: (a) (i) aheavy chain constant region selected from the group consisting of humanIgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂, (ii) an IgG₁ heavy chainconstant region comprising an N297A mutation, numbered according to theEU numbering system, (iii) a heavy chain constant region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:47-58, or (iv) a heavy chain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 81, 82, 85, 86, 115, 117, 119,121, and 127; (b) (i) a light chain constant region selected from thegroup consisting of human kappa light chain and human lambda lightchain, (ii) a light chain constant region comprising the amino acidsequence of SEQ ID NO: 93, or (iii) a light chain comprising the aminoacid sequence of SEQ ID NO: 97; or (c) a heavy chain and a light chaincomprising the amino acid sequences selected from the group consistingof SEQ ID NOs: 81 and 97; 82 and 97; 85 and 97; 86 and 97; 115 and 97;117 and 97; 119 and 97; 121 and 97; and 127 and 97, respectively.
 7. Theisolated polynucleotide of claim 1, wherein the antibody comprises: aheavy chain comprising the amino acid sequence of SEQ ID NO: 85; and alight chain comprising the amino acid sequence of SEQ ID NO:
 97. 8. Theisolated polynucleotide of claim 1, wherein the antibody comprises: aheavy chain comprising the amino acid sequence of SEQ ID NO: 115, and alight chain comprising the amino acid sequence of SEQ ID NO:
 97. 9. Theisolated polynucleotide of claim 1, wherein the polynucleotide furtherencodes a TGFβ-binding moiety selected from the group consisting of: (i)an extracellular domain of a human TGFβ receptor; and (ii) an antibodythat specifically binds to TGFβ.
 10. The isolated polynucleotide ofclaim 9, wherein the extracellular domain of the human TGFβ receptorcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 109-112.
 11. The isolated polynucleotide of claim 9, whereinthe extracellular domain of the human TGFβ receptor comprises the aminoacid sequence of SEQ ID NO:
 111. 12. The isolated polynucleotide ofclaim 9, wherein the polynucleotide further encodes a peptide linkercomprising an amino acid sequence that is at least 95% identical to anamino acid sequence selected from the group consisting of SEQ ID NOs:103-108.
 13. The isolated polynucleotide of claim 1, wherein theantibody comprises a heavy chain comprising an amino acid sequence thatis at least 95% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 115, 117, 119, and 121, and/or a lightchain comprising an amino acid sequence that is at least 95% identicalto the amino acid sequence of SEQ ID NO:
 97. 14. The isolatedpolynucleotide of claim 1, wherein the polynucleotide further encodes aVEGF-binding moiety selected from the group consisting of: (i) anextracellular domain of a human VEGF receptor; and (ii) an antibody thatspecifically binds to VEGF.
 15. The isolated polynucleotide of claim 14,wherein the extracellular domain of the human VEGF receptor comprisesthe amino acid sequence of SEQ ID NO:
 122. 16. The isolatedpolynucleotide of claim 14, wherein the polynucleotide further encodes apeptide linker comprising an amino acid sequence that is at least 95%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 103-108.
 17. The isolated polynucleotide of claim 1,wherein the antibody comprises a heavy chain comprising an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO: 127, and/or a light chain comprising an amino acid sequencethat is at least 95% identical to the amino acid sequence of SEQ ID NO:97.
 18. A vector comprising: (a) the polynucleotide of claim 1; (b) thepolynucleotide of claim 1 and a second polynucleotide encoding anextracellular domain of a human TGFβ receptor; or (c) the polynucleotideof claim 1 and a second polynucleotide encoding an extracellular domainof a human VEGF receptor.
 19. A recombinant host cell comprising thepolynucleotide of claim
 1. 20. A recombinant host cell comprising afirst polynucleotide encoding a heavy chain variable region or a heavychain of an antibody and a second polynucleotide encoding a light chainvariable region or a light chain of the antibody, wherein the antibodycomprises: a heavy chain variable region comprising the CDRH1, CDRH2,and CDRH3 amino acid sequences of the heavy chain variable region of SEQID NO: 27; and a light chain variable region comprising the CDRL1,CDRL2, and CDRL3 amino acid sequences of the light chain variable regionof SEQ ID NO:
 37. 21. The recombinant host cell of claim 20, wherein:(a) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the CDRH1,CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences,respectively, set forth in SEQ ID NOs: 2, 5, 7, 8, 10, and 12; (b) theheavy chain variable region comprises the amino acid sequence set forthin SEQ ID NO: 27 and the light chain variable region comprises the aminoacid sequence set forth in SEQ ID NO: 37; (c) the heavy chain comprisesthe amino acid sequence of SEQ ID NO: 85 or 115; and the light chaincomprises the amino acid sequence of SEQ ID NO: 97; and/or (d) therecombinant host cell further comprises a polynucleotide encoding anextracellular domain of a human TGFβ or VEGF receptor.
 22. Therecombinant host cell of claim 20, wherein the antibody comprises aheavy chain comprising an amino acid sequence that is at least 95%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 115, 117, 119, 121, and 127, and/or a light chaincomprising an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:
 97. 23. A lipid nanoparticlecomprising the polynucleotide of claim
 1. 24. A method of producing anantibody, the method comprising culturing the host cell of claim 19under suitable conditions so that the polynucleotide is expressed andthe antibody is produced.
 25. A method of producing an antibody, themethod comprising culturing the host cell of claim 20 under suitableconditions so that the polynucleotide is expressed and the antibody isproduced.